Noncompetitive Determination of Small Analytes by Sandwich-Type Lateral Flow Assay Based on an Aptamer Kissing Complex.

Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a noncompetitive manner by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and the capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.

Understanding Parahydrogen Hyperpolarized Urine Spectra: The Case of Adenosine Derivatives.

Parahydrogen hyperpolarization has emerged as a promising tool for sensitivity-enhanced NMR metabolomics. It allows resolution and quantification of NMR signals of certain classes of low-abundance metabolites that would otherwise be undetectable. Applications have been implemented in pharmacokinetics and doping drug detection, demonstrating the versatility of the technique. Yet, in order for the method to be adopted by the analytical community, certain limitations have to be understood and overcome. One such question is NMR signal assignment. At present, the only reliable way to establish the identity of an analyte that gives rise to certain parahydrogen hyperpolarized NMR signals is internal standard addition, which can be laborious. Herein we show that analogously to regular NMR metabolomics, generating libraries of hyperpolarized analyte signals is a viable way to address this limitation. We present hyperpolarized spectral data of adenosines and give an early example of identifying them from a urine sample with the small library. Doing so, we verify the detectability of a class of diagnostically valuable metabolites: adenosine and its derivatives, some of which are cancer biomarkers, and some are central to cellular energy management (e.g., ATP).

Determination of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine in urine using solvent-modified micellar electrokinetic chromatography.

A new approach for direct determination of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and methylthioadenosine (MTA) in urine was developed based on MEKC by using SDS modified with isobutanol in the presence of PEG-300. Analytes were first extracted with grafted phenylborononic acid. Using a 50 µm internal diameter silica capillary of 32 cm total length filled with 0.05 M SDS, 0.05 M H3 PO4 , 5% (v/v) isobutanol, and 10% (v/v) PEG-300, LOQ of 0.15 µM for SAM and SAH, and 0.2 µM for MTA was reached. Accuracy was 92% for MTA, 109% for SAH, and 105% for SAM, intra- and interday imprecision were <2.5 and ≤3%, respectively. The total time of analysis for one sample was 10 min. Analysis of 30 urine samples from healthy volunteers showed that the median SAM and SAH levels were 12.1 and 0.73 µM, respectively. MTA levels, which were determined in urine for the first time (according to our data), were 0.43 µM, and these values correlated well with the SAM level (r = 0.748, p < 0.01).

Development of a gas chromatography-mass spectrometry method for breast cancer diagnosis based on nucleoside metabolomes 1-methyl adenosine, 1-methylguanosine and 8-hydroxy-2'-deoxyguanosine.

Metabolomes are small molecule metabolites (<1000 Da) produced by cellular processes. Metabolomes are close counterparts to the genome, transcriptome and proteome. The aim of this study was to develop a method to detect and quantify candidate nucleoside metabolomes 1-methyl adenosine (1-MA), 1-methylguanosine (1-MG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the urine of patients with breast cancer using gas chromatography-mass spectrometry (GC-MS). The method was applied to urine specimens from patients with breast cancer (n = 56) and benign breast tumors (n = 22), as well as from healthy females (n = 20). The relative standard deviations of precision and repeatability analysis were <10%, and recoveries ranged from 88.5 to 105.6%. Limits of detection were 0.014, 0.012, and 0.018 mg/L for 1-MA, 1-MG and 8-OHdG, respectively. The lower limits of quantitation were 0.056, 0.048 and 0.072 mg/L, respectively. There were significant differences in concentrations of candidate metabolomes between patients with cancer and the healthy individuals, especially for those in the early stages of the disease (p < 0.001). No significant differences were observed between the benign and healthy groups. In conclusion, a reliable GC-MS method for the detection and quantification of 1-MA, 1-MG, and 8-OHdG metabolomes in urine has been developed.

Reduced levels of modified nucleosides in the urine of autistic children. Preliminary studies.

The aim of this study was to investigate and compare the levels of concentration of modified nucleosides in the urine of autistic and healthy children. The compounds have never been analyzed before. The levels of nucleosides in the urine of both groups were determined by validated high performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. Chromatographic separation was achieved with HILIC column and tubercidin was used as the internal standard for the quantification of urinary nucleosides. The within run accuracy and precision ranged from 89 to 106% and from 0.8% to 4.9%, respectively. Lower levels of O-methylguanosine, 7-methylguanosine, 1-methyladenosine, 1-methylguanine, 7-methylguanine and 3-methyladenine in the urine of 22 children with autism, aged 3 to 16 were observed. The differences were not observed in 20 healthy volunteers, in a similar age group. These findings show that modified nucleosides there are metabolic disturbances and nutritional deficiencies in autistic children.

A split aptamer-labeled ratiometric fluorescent biosensor for specific detection of adenosine in human urine.

A dual-emission ratiometric fluorometric aptasensor is presented for highly specific detection of adenosine. An adenosine binding aptamer (ABA) was split into two halves (termed as ABA1 and ABA2). ABA1 was covalently bound to blue-emitting carbon dots (with excitation/emission maxima at 365/440 nm) as responsive fluorophore (referred to as ABA1-CDs). ABA2 was linked to red-emitting silica-coated CdTe quantum dots (with excitation/emission maxima at 365/613 nm) acting as internal reference and referred to as ABA2-QDs@SiO2. Upon addition of graphene oxide, the fluorescence of ABA1-CDs is quenched. After subsequent addition of ABA2-QDs@SiO2 and different amounts of adenosine, the blue fluorescence is recovered and causes a color change from red to royal blue. The method represents a ratiometric turn-on assay for visual, colorimetric and fluorometric determination of adenosine. The limit of detection is as low as 2.4 nM in case of ratiometric fluorometry. The method was successfully applied to the determination of adenosine in (spiked) human urine. Recoveries range from 98.8% to 102%. Graphical abstract Adenosine binding aptamer1-carbon dots (ABA1-CDs) can absorb on graphene oxide (GO) via π stacking. This causes fluorescence to be quenched by fluorescence resonance energy transfer (FRET). After addition of ABA2-silica-coated quantum dots (ABA2-QDs@SiO2) and adenosine, binding of adenosine to two pieces of aptamers forms a complex (ABA1-CD/adenosine/ABA2-QD@SiO2) which dissociates from GO. As a result, fluorescence is recovered.

Urinary adenosine excretion in type 1 diabetes.

In experimental models of diabetes, augmented sodium-glucose cotransport-2 (SGLT2) activity diminishes sodium (Na+) delivery at the macula densa. As a result, less vasoconstrictive adenosine is generated, leading to afferent arteriolar vasodilatation and hyperfiltration. The measurement and significance of urinary adenosine in humans has not been examined extensively in states of renal hemodynamic impairment like that of diabetes. Our aim was to validate a method for urine adenosine quantification in humans and perform an exploratory post hoc analysis to determine whether urinary adenosine levels change dynamically in response to natriuresis in patients with type 1 diabetes (T1D) before and after treatment with the SGLT2 inhibitor (SGLT2i) empagliflozin. We hypothesized that SGLT2i, which reduces renal hyperfiltration through increased Na+ delivery to the macula densa, would increase urinary adenosine excretion. Urine adenosine corrected for creatinine was measured using our validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in 40 healthy participants and 40 patients with T1D. In the T1D cohort, measurements were performed during clamped euglycemic and hyperglycemic conditions before and following 8 wk of SGLT2i therapy. Urinary adenosine was detectable in healthy subjects (0.32 ± 0.11 µmol/mmol Cr) and patients with T1D. In response to SGLT2i, urine adenosine increased during clamped hyperglycemia (0.40 ± 0.11 vs. 0.45 ± 0.12 µmol/mmol Cr, P = 0.005). Similar trends were observed during clamped euglycemia (P = 0.08). In conclusion, SGLT2i increases urinary adenosine excretion under clamped hyperglycemic conditions in patients with T1D. The potentially protective role of SGLT2i against glomerular hyperfiltration and its mediation by adenosine in diabetes merits further study.

Adenosine kinase deficiency: expanding the clinical spectrum and evaluating therapeutic options.

BACKGROUND: Adenosine kinase deficiency is a recently described defect affecting methionine metabolism with a severe clinical phenotype comprising mainly neurological and hepatic impairment and dysmorphism. METHODS: Clinical data of 11 additional patients from eight families with adenosine kinase deficiency were gathered through a retrospective questionnaire. Two liver biopsies of one patient were systematically evaluated. RESULTS: The main clinical symptoms are mild to severe liver dysfunction with neonatal onset, muscular hypotonia, global developmental retardation and dysmorphism (especially frontal bossing). Hepatic involvement is not a constant finding. Most patients have epilepsy and recurrent hypoglycemia due to hyperinsulinism. Major biochemical findings are intermittent hypermethioninemia, increased S-adenosylmethionine and S-adenosylhomocysteine in plasma and increased adenosine in urine. S-adenosylmethionine and S-adenosylhomocysteine are the most reliable biochemical markers. The major histological finding was pronounced microvesicular hepatic steatosis. Therapeutic trials with a methionine restricted diet indicate a potential beneficial effect on biochemical and clinical parameters in four patients and hyperinsulinism was responsive to diazoxide in two patients. CONCLUSION: Adenosine kinase deficiency is a severe inborn error at the cross-road of methionine and adenosine metabolism that mainly causes dysmorphism, brain and liver symptoms, but also recurrent hypoglycemia. The clinical phenotype varies from an exclusively neurological to a multi-organ manifestation. Methionine-restricted diet should be considered as a therapeutic option.

Increased levels of adenosine and ecto 5'-nucleotidase (CD73) activity precede renal alterations in experimental diabetic rats.

The pathogenesis of diabetic nephropathy (DN) has not been clearly established, making diagnosis and patient management difficult. Recent studies using experimental diabetic models have implicated adenosine signaling with renal cells dysfunction. Therefore, the study of the biochemical mechanisms that regulate extracellular adenosine availability during DN is of emerging interest. Using streptozotocin-induced diabetic rats we demonstrated that urinary levels of adenosine were early increased. Further analyses showed an increased expression of the ecto 5'-nucleotidase (CD73), which hydrolyzes AMP to adenosine, at the renal proximal tubules and a higher enzymatic activity in tubule extracts. These changes precede the signs of diabetic kidney injury recognized by significant proteinuria, morphological alterations and the presence of the renal fibrosis markers alpha smooth muscle actin and fibronectin, collagen deposits and thickening of the glomerular basement membrane. In the proximal tubule cell line HK2 we identified TGF-ß as a key modulator of CD73 activity. Importantly, the increased activity of CD73 could be screened in urinary sediments from diabetic rats. In conclusion, the increase of CD73 activity is a key component in the production of high levels of adenosine and emerges as a new tool for the early diagnosis of tubular injury in diabetic kidney disease.

Gold-nanoparticle extraction and reversed-electrode-polarity stacking mode combined to enhance capillary electrophoresis sensitivity for conjugated nucleosides and oligonucleotides containing thioether linkers.

We present a capillary electrophoresis method for determining two different C8-conjugated deoxyadenosines, and for oligonucleotides containing them, in which a psoralen or an acridine molecule is bonded to the base via a short alkyl chain containing sulfur ethers at both ends. The sensitivity of the micellar electrokinetic chromatography (MEKC) method was increased by using two preconcentration techniques, micro solid-phase extraction (µSPE) followed by reversed-electrode-polarity stacking mode (REPSM). Variables that affect the efficiency of the extraction in µSPE and preconcentration by REPSM, including the type and volume of extraction nanoparticle, concentration, and injection time, were investigated. Under the optimum conditions, enrichment factors obtained were in the range 360-400. The limits of detection (LODs) at a signal-to-noise ratio of 3 ranged from 2 to 5 nmol L(-1). The relative recoveries of labelled adenosines from water samples were 95-103%. The proposed method provided high enrichment factors and good precision and accuracy with a short analysis time. On the basis of the advantages of simplicity, high selectivity, high sensitivity, and good reproducibility, the proposed method may have great potential for biochemical applications.

Gender-related effects on urine L-cystine metastability.

Cystinuria is an autosomal recessive disease that causes L-cystine precipitation in urine and nephrolithiasis. Disease severity is highly variable; it is known, however, that cystinuria has a more severe course in males. The aim of this study was to compare L-cystine metastability in first-morning urine collected from 24 normal female and 24 normal male subjects. Samples were buffered at pH 5 and loaded with L-cystine (0.4 and 4 mM final concentration) to calculate the amount remaining in solution after overnight incubation at 4 °C; results were expressed as Z scores reflecting the L-cystine solubility in each sample. In addition, metabolomic analyses were performed to identify candidate compounds that influence L-cystine solubility. L-cystine solubility Z score was +0.44 ± 1.1 and -0.44 ± 0.70 in female and male samples, respectively (p < 0.001). Further analyses showed that the L-cystine solubility was independent from urine concentration but was significantly associated with low urinary excretion of inosine (p = 0.010), vanillylmandelic acid (VMA) (p = 0.015), adenosine (p = 0.029), and guanosine (p = 0.032). In vitro L-cystine precipitation assays confirmed that these molecules induce higher rates of L-cystine precipitation in comparison with their corresponding dideoxy molecules, used as controls. In silico computational and modeling analyses confirmed higher binding energy of these compounds. These data indicate that urinary excretion of nucleosides and VMA may represent important factors that modulate L-cystine solubility and may represent new targets for therapy in cystinuria.

Adenosine A(2B) receptor-mediated VEGF induction promotes diabetic glomerulopathy.

Diabetic nephropathy ranks as the most devastating kidney disease worldwide. It characterizes in the early onset by glomerular hypertrophy, hyperfiltration and mesangial expansion. Experimental models show that overproduction of vascular endothelial growth factor (VEGF) is a pathogenic condition for podocytopathy; however the mechanisms that regulate this growth factor induction are not clearly identified. We determined that the adenosine A(2B) receptor (A(2B)AR) mediates VEGF overproduction in ex vivo glomeruli exposed to high glucose concentration, requiring PKCα and Erk1/2 activation. The glomerular content of A(2B)AR was concomitantly increased with VEGF at early stages of renal disease in streptozotocin-induced diabetic rats. Further, in vivo administration of an antagonist of A(2B)AR in diabetic rats blocked the glomerular overexpression of VEGF, mesangial cells activation and proteinuria. In addition, we also determined that the accumulation of extracellular adenosine occurs in glomeruli of diabetic rats. Correspondingly, raised urinary adenosine levels were found in diabetic rats. In conclusion, we evidenced that adenosine signaling at the onset of diabetic kidney disease is a pathogenic event that promotes VEGF induction.

Parahydrogen hyperpolarization of minimally altered urine samples for sensitivity enhanced NMR metabolomics.

Parahydrogen hyperpolarization has been shown to enhance NMR sensitivity in urine analysis by several orders of magnitude if urine samples are prepared by solid phase extraction (SPE). We present a different approach, developed for minimal sample alteration before analysis. Removing SPE from the workflow allows to retain a wider range of metabolites and paves the way towards more universal hyperpolarized NMR metabolomics of low abundance metabolites.

Disposition and metabolism of ticagrelor, a novel P2Y12 receptor antagonist, in mice, rats, and marmosets.

Ticagrelor is a reversibly binding and selective oral P2Y(12) antagonist, developed for the prevention of atherothrombotic events in patients with acute coronary syndromes. The disposition and metabolism of [(14)C]ticagrelor was investigated in mice, rats, and marmosets to demonstrate that these preclinical toxicity species showed similar metabolic profiles to human. Incubations with hepatocytes or microsomes from multiple species were also studied to compare with in vivo metabolic profiles. The routes of excretion were similar for both oral and intravenous administration in mice, rats, and marmosets with fecal excretion being the major elimination pathway accounting for 59 to 96% of the total radioactivity administered. Urinary excretion of drug-related material accounted for only 1 to 15% of the total radioactivity administered. Milk samples from lactating rats displayed significantly higher levels of total radioactivity than plasma after oral administration of ticagrelor. This demonstrated that ticagrelor and/or its metabolites were readily transferred into rat milk and that neonatal rats could be exposed to ticagrelor-related compounds via maternal milk. Ticagrelor and active metabolite AR-C124910 (loss of hydroxyethyl side chain) were the major components in plasma from all species studied and similar to human plasma profiles. The primary metabolite of ticagrelor excreted in urine across all species was an inactive metabolite, AR-C133913 (loss of difluorophenylcyclopropyl group). Ticagrelor, AR-C124910, and AR-C133913 were the major components found in feces from the three species examined. Overall, in vivo metabolite profiles were qualitatively similar across all species and consistent with in vitro results.

Identification of major metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside by LC/MS/MS.

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.

Determination of urinary adenosine using resonance light scattering of gold nanoparticles modified structure-switching aptamer.

A novel sensitive method has been developed for the detection of adenosine (AD) in human urine by using enhanced resonance light scattering (RLS). This method is based on the specific recognition and signal amplification of adenosine aptamer (Apt) coupled with gold nanoparticles (GNPs) via G-quartet-induced nanoparticle assembly, which was fabricated by triggering a structure switching of the 3' terminus G-rich sequence and aptamer duplex. RLS signal linearly correlated with the concentration of adenosine over the range of 6-115nM. The limit of detection (LOD) for adenosine is 1.8nM with relative standard deviations (RSD) of 2.90-4.80% (n=6). The present method has been successfully applied to determination of adenosine in real human urine, and the obtained results were in good agreement with those obtained by the HPLC method. Our investigation shows that the combination of the excellent selectivity of aptamer with the high sensitivity of the RLS technique could provide a promising potential for aptamer-based small molecule detection, and be beneficial in extending the application of RLS.

Adenylosuccinate lyase deficiency in a Malaysian patient, with novel adenylosuccinate lyase gene mutations.

Most cases of adenylosuccinate lyase (ADSL OMIM 103050) deficiency reported to date are confined to the various European ethnic groups. We report on the first Malaysian case of ADSL deficiency, which appears also to be the first reported Asian case. The case was diagnosed among a cohort of 450 patients with clinical features of psychomotor retardation, global developmental delay, seizures, microcephaly and/or autistic behaviour. The patient presented with frequent convulsions and severe myoclonic jerk within the first few days of life and severe psychomotor retardation. The high performance liquid chromatography (HPLC) profile of the urine revealed the characteristic biochemical markers of succinyladenosine (S-Ado) and succinyl-aminoimidazole carboximide riboside (SAICAr). The urinary S-Ado/SAICAr ratio was found to be 1.02 (type I ADSL deficiency). The patient was compound heterozygous for two novel mutations, c.445C > G (p.R149G) and c.774_778insG (p.A260GfsX24).

Biocatalytic growth of gold agglomerates on an electrode for aptamer-based electrochemical detection.

Here we describe the biocatalytic growth of high-density gold agglomerates on a gold electrode surface to form a carrier for aptamer probe immobilization. The present approach provides a simple strategy to promote the seed-mediated deposition of Au from AuCl(4)(-) onto surface-attached 12 nm diameter Au nanoparticles (AuNPs) in the presence of reductive coenzyme and surfactant. The growth process was studied by electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). This nanostructured platform is effective and prospective toward the aptamer probe immobilization. For the nice performance of enhanced substrate, the aptamer-sensing interface showed excellent applicability under the investigations such as alternating current voltammetry (ACV) and surface-enhanced Resonance Raman scattering (SERRS) spectra.

Applicability of Supercritical fluid chromatography-Mass spectrometry to metabolomics. II-Assessment of a comprehensive library of metabolites and evaluation of biological matrices.

In this work, the impact of biological matrices, such as plasma and urine, was evaluated under SFCHRMS in the field of metabolomics. For this purpose, a representative set of 49 metabolites were selected. The assessment of the matrix effects (ME), the impact of biological fluids on the quality of MS/MS spectra and the robustness of the SFCHRMS method were each taken into consideration. The results have highlighted a limited presence of ME in both plasma and urine, with 30% of the metabolites suffering from ME in plasma and 25% in urine, demonstrating a limited sensitivity loss in the presence of matrices. Subsequently, the MS/MS spectra evaluation was performed for further peak annotation. Their analyses have highlighted three different scenarios: 63% of the tested metabolites did not suffer from any interference regardless of the matrix; 21% were negatively impacted in only one matrix and the remaining 16% showed the presence of matrix-belonging compounds interfering in both urine and plasma. Finally, the assessment of retention times stability in the biological samples, has brought into evidence a remarkable robustness of the SFCHRMS method. Average RSD (%) values of retention times for spiked metabolites were equal or below 0.5%, in the two biological fluids over a period of three weeks. In the second part of the work, the evaluation of the Sigma Mass Spectrometry Metabolite Library of Standards containing 597 metabolites, under SFCHRMS conditions was performed. A total detectability of the commercial library up to 66% was reached. Among the families of detected metabolites, large percentages were met for some of them. Highly polar metabolites such as amino acids (87%), nucleosides (85%) and carbohydrates (71%) have demonstrated important success rates, equally for hydrophobic analytes such as steroids (78%) and lipids (71%). On the negative side, very poor performance was found for phosphorylated metabolites, namely phosphate-containing compounds (14%) and nucleotides (31%).

An in-tube aptamer/gold nanoparticles coated capillary solid-phase microextraction for separation of adenosine in serum and urine samples.

As an endogenous nucleoside, adenosine was significant for the diagnosis and treatment of some diseases, such as schizophrenia. However, due to the complicated matrix interference, it was difficult to monitor trace or ultra-trace adenosine directly in bio-samples. In this contribution, a novel in-tube SPME technique based on aptamer/Au nanoparticles coated open tubular fused-silica capillary was established to separate and enrich adenosine in bio-samples with high affinity. Therefore, a uniform and dense AuNPs layer was coated on the inner surface of the open tubular capillary, and then adenosine aptamer was immobilized on AuNPs with a high capacity of 2.44 µg per 27-cm capillary. As a result, the capillary shown high selectivity to adenosine with a selectivity factor of 14.4 when compared with the scrambled aptamer/AuNPs coated capillary. Also, the extraction amount of adenosine was 2.8-24.8 times higher than those of its structural analogs and contrast, such as guanosine, uridine, cytidine, thymidine, and toluic acid. After the optimization of extraction conditions, the aptamer/AuNPs coated in-tube SPME-HPLC method was developed for the adenosine assay with the linear range of 0.002-0.100 µg mL-1 and the detection limit of 0.45 ng mL-1. Subsequently, the approach was applied for trace adenosine monitoring in human serum and urine samples. It showed a strong performance of reducing matrix interference and improving sensitivity, and the spiking recoveries of 89.9-92.6% and 91.1-94.5% were achieved respectively.