nM In Urine Research Articles

Extracellular RNA splice events in cerebrospinal fluid as candidate biomarkers of myotonic dystrophy type 1 (N4.004)

<h3>Objective:</h3> To evaluate extracellular RNA (exRNA) in cerebrospinal fluid (CSF) as a source of CNS-derived splice events that may serve as biomarker of DM1 disease activity. <h3>Background:</h3> Alternative splicing is mis-regulated in post-mortem DM1 central nervous system (CNS) tissue. In past few years Cerebrospinal Fluid (CSF) biomarkers have been used to characterize disease severity and prognosis of the disease and helpful tool for to measure changes after treatment. exRNA in CSF provides a source of CNS-derived splice events in living individuals. <h3>Design/Methods:</h3> We examined fresh CSF samples from unaffected controls (N=14) from the MGH CSF biobank and DM1 individuals with mild CNS involvement (N=2). To separate EVs from cells contained in CSF, we used low speed centrifugation followed by filtration of the supernatant. EV size and concentration were measured using microscopy and particle tracking analysis software (NanoSight). We extracted exRNA from the EV pellet, produced cDNA, and quantified gene expression and splice events (% exon inclusion) by droplet digital PCR. <h3>Results:</h3> Mean particle diameter is about 175 nm in CSF and serum vs. 220 nm in urine. In CSF cells, total RNA content is 10 – 100-fold higher than exRNA. Normalized expression of both <i>DMPK</i> and <i>CNBP</i> was 50 – 60% higher in CSF exRNA than in CSF cells. Splicing is significantly different in CSF exRNA vs CSF cells. Alternative splicing of GOLGA4 exon 23 inclusion was 65% in DM1 and 40% in UA (P&lt;0.01). Differential splicing of NUMA1, NCOR2 and MBNL2 was non-significant between groups. <h3>Conclusions:</h3> Quantification of CNS-derived alternative splice products in CSF exRNA is feasible. Removal of leukocytes and erythrocytes normally found in CSF will enhance accurate quantification of CNS-derived splice events. GOLGA4 is a candidate biomarker of early CNS involvement in DM1, while NUMA1, NCOR2, and MBNL2 splice events are candidate biomarkers of disease severity. <b>Disclosure:</b> Dr. Kumari has nothing to disclose. Mr. Hu has nothing to disclose. Mr. Sizemore has nothing to disclose. Ms. Sullivan has nothing to disclose. Mr. McKee has nothing to disclose. Mr. Conquest has nothing to disclose. Dr. Wheeler has nothing to disclose.

SnO2 quantum dots-functionalized Ti3C2 MXene nanosheets for electrochemical determination of dopamine in body fluids.

Anovel SnO2 quantum dots (SnO2QDs)-functionalized Ti3C2 MXene nanocomposite was prepared via in situ synthesis method, resulting in well-regulated the nucleation and growth of SnO2QDs to evenly distribute onto MXene nanosheets. Ultra-small size SnO2QDs decorated on the surface of Ti3C2 MXene nanosheets can effectively prevent the restacking of MXene and remarkably increase the electroactive surface area of theelectrode, which can further increase electrocatalytic activity toward dopamine. Then, an ultrasensitive electroanalytical method based on SnO2QDs-functionalized Ti3C2 MXene nanocomposite for dopamine monitoring was developed, and the effects of experimental condition were investigated systematically. Under optimized conditions, the prepared sensor presented a linear dependence for dopamine in the concentration range from 0.004 to 8.0µM with the detection limit of 2.0nM (S/N = 3). Moreover, it selectively perceived dopamine in presence of physiological interferents in urine and serum samples with excellent linearities (correlation coefficients higher than 0.9920). The relative recoveries were in the range 97.67-105.3% and 103.0-106.8%, while the limits of quantitation were 10.12nM and 9.62nM in urine and serum sample, respectively, demonstrating the method suitability for dopamine sensing and being envisioned as a promising candidate for neurotransmitter monitoring in biological diagnosis.

A simple AIEgen photosensitizer with cucurbit[7]uril selective detection amantadine and application in mitochondrion imaging

In this work, an AIEgen photosensitizer TPM is synthesized and simple strategy for detecting amantadine (AM) in aqueous solutions and urine is proposed by combining cucurbit[7]uril (CB[7]) with TPM. High emission from the aqueous solution is obtained by the interaction of TPM with CB[7], which formed CB[7]@TPM. The 1H NMR spectra and isothermal titration calorimetry (ITC) results revealed that TPM is encapsulated in CB[7] in both aqueous solution and urine to form a stable 1:1 host–guest inclusion complex of CB[7]@TPM. When AM added to a CB[7]@TPM solution, strong interconnections formed, which occupied the cavity of CB[7] and formed a complex, CB[7]@AM. Based on the significant quenching of the supramolecular complex’s photoluminescence (PL) intensity, a fluorescence method with high sensitivity and selectivity is developed for the determination of AM in aqueous solutions and urine. The limits of detection (LOD) for AM are determined to be 72 nM in aqueous solution and 80.9 nM in urine. Importantly, due to its amphiphilicity and cationic nature, the TPM can specifically stain the mitochondria in living cells and acted as an efficient photosensitizer for killed cancer cells.

Electrochemical detection of triamterene in human urine using boron-doped diamond electrodes

Urine is one of the most used biological fluids for screening drug delivery and the resultant metabolites. In sports, the use of diuretics such as triamterene is considered a violation of anti-doping rules and is stipulated to be present at less than 79 nM in urine by the World Anti-Doping Agency (WADA). It is therefore important to develop effective rapid and low-cost tests for this diuretic. Here we apply electrochemical analysis using boron-doped diamond (BDD) electrodes, which have superior properties such as low background current, a wide potential window, and high resistance to deactivation. Since real urine samples show clear oxidation current peaks in the potential range more positive than 0.5 V (vs. Ag/AgCl) due to the presence of bio-components such as protein, uric acid, and ascorbic acid, to detect triamterene effectively, the electrochemical protocol was optimized towards a potential range where the other components have limited effect. Our results show that reduced triamterene exhibits an oxidation peak at 0.1 V (vs. Ag/AgCl) in 0.1 M phosphate buffer (PB) and at 0.2 V (vs. Ag/AgCl) in pooled human urine. The peak current value increased according to the triamterene concentration. The limit of detection (LOD) was 3.15 nM in the PB and 7.80 nM in pooled human urine. Finally, triamterene detection was attempted in individual urine samples. Triamterene was electrochemically detectable in individual urine samples, excluding urine samples containing an excess amount of ascorbic acid. The limit of detection (LOD) in individual urine samples was determined to be 20.8 nM.

Ultrasensitive Detection of Tetracycline Using Boron and Nitrogen Co-Doped Graphene Quantum Dots from Natural Carbon Source as the Paper-Based Nanosensing Probe in Difference Matrices.

Herein, the boron and nitrogen co-doped 0-dimensional graphene quantum dots (B,N-GQDs) with high quantum yield (QY) were synthesized via microwave-assisted hydrothermal method at 170 °C for 20 min using fresh passion fruit juice and boric acid as the starting materials. The 3–6 layers of B,N-GQDs with mean particle size of 9 ± 1 nm were then used for ultra-sensitive and selective detection of tetracycline in aqueous and biological media. The hybridization of boron and nitrogen atoms into the GQD structures increases the intensity of electronegative, resulting in the enhancement of QY to 50 ± 1%. The B,N-GQDs show their excellent analytical performance on tetracycline determination after 2 min of reaction under an optimal condition at pH 5. The linear range of 0.04–70 µM and with limits of detection (LOD) of 1 nM in phosphate buffer saline (PBS), 1.9 nM in urine and 2.2 nM in human serum are obtained. Moreover, the high selectivity of tetracycline by B,N-GQDs over the other 23 interferences is observed. The π-π interaction and electron donor-acceptor principle play pivotal roles in enhancing the ultra-sensitivity and selectivity of B,N-GQDs toward TC detection. Moreover, the B, N-GQD based paper nanosensor exhibits an excellent analytical performance on visual detection of 0.1–30 µM TC in human serum. Results of this study clearly indicate the feasibility of synthesis of B,N-GQDs derived from passion fruit juice for ultrasensitive tetracycline detection, which can open an avenue to use natural products for the preparation of environmentally benign and biocompatible carbon nanomaterials for highly sensitive detection of drugs, antibiotics, organic compounds and biomarkers.

Trapping Methylglyoxal by Myricetin and Its Metabolites in Mice.

Trapping of methylglyoxal (MGO) has been determined to be one of the potential mechanisms for dietary polyphenols to prevent chronic diseases. In this study, myricetin was demonstrated to efficiently trap MGO to generate mono- and di-MGO adducts under in vitro conditions. Furthermore, the mono- and di-MGO adducts of myricetin were detected in urine and fecal samples collected from myricetin-treated mice based on LC-MS analysis. More importantly, the mono-MGO adducts of the mono- and di-methylated myricetin were also found in these mouse samples. Further dose-dependent studies demonstrated that myricetin and its methylated metabolites significantly trapped MGO in a dose-dependent manner with the 400 mg/kg dose having the highest trapping efficacy (mono-MGO-myricetin: 272.0 ± 90.9 nM in urine and 1.05 ± 0.67 μg/g in feces; mono-MGO-mono-Me-myricetin: 135.2 ± 77.6 nM in urine and 1.16 ± 0.65 μg/g in feces; and mono-MGO-di-Me-myricetin: 17.0 ± 5.9 nM in urine and 0.19 ± 0.04 μg/g in feces) compared to the 100 and 200 mg/kg doses. In conclusion, this study demonstrates for the first time the in vivo trapping efficacy of myricetin, suggesting that intake of myricetin-containing foods has the potential to scavenge MGO in vivo and to prevent MGO-induced harmful effects to human health.

A ratiometric fluorescent probe for sensitive determination of the important glycopeptide antibiotic vancomycin.

A novel sensitive and selective probe for the important antibiotic vancomycin (Van) has been synthesized by integrating a coumarin and a fluorescein as dual fluorescence reporters and a Van binding peptide D-Ala-D-Ala. Only weak green fluorescence was initially observed, which was mostly attributed to fluorescence self-quenching induced by fluorophore stacking. Upon the binding of Van with the D-Ala-D-Ala peptide, the fluorescence turned on, probably due the disaggregation of fluorophores. The intensity ratio of the dual emission bands I519/I446 exhibited an excellent linear relationship with the concentration of Van increasing from 0-20 μM in synthetic urine. The lowest detection limit was calculated to be 92.8 nM in urine, which made the probe applicable in clinically relevant concentration ranges. The synthetic probe has also shown the potential for Van detection in human serum. More interestingly, this probe has been successfully applied for in vivo imaging of Van in zebrafish. Graphical Abstract.

Direct and sensitive determination of trypsin in human urine using a water-soluble signaling fluorescent molecularly imprinted polymer nanoprobe

Abstract A molecularly imprinted polymer (MIP) for biosensing trypsin is prepared by incorporating a benzamidine-based signaling fluorescent monomer during its synthesis. Binding to trypsin results in a 100-fold fluorescence enhancement when the MIP is excited with UV light. The assay can be performed by simply mixing a small amount of water-soluble MIP (100 μg) and trypsin together in a quartz cuvette, followed by a direct read-out on a spectrofluorimeter. There is no need to separate the free from the bound trypsin. The limit of quantification is 50 nM in phosphate buffer and 210 nM in urine. The aqueous MIP is prepared by a solid-phase synthesis method on glass-beads functionalized with a metal chelate to immobilize trypsin via its surface histidine. The active site of the enzyme is left free for binding to the benzamidine moiety of the fluorescent monomer, resulting in a sensitive MIP for probing the enzyme’s active site. Incorporation of a thermoresponsive monomer, N-isopropyacrylamide, in the polymerization mixture, yields thermoresponsive MIP nanoparticles that are released from the support by a simple temperature change, generating template-free polymers. The MIPs are endowed with improved binding site homogeneity since all binding sites have the same orientation. Their size is ∼70 nm and the dissociation constant (Kd) of the MIP-trypsin complex is 237 nM, with little or no cross-reactivity with other proteins, including serine proteases.

Recognition and quantification of HSA: A fluorescence probe across α-helices of site I and site II

We reported a new fluorescence probe based on Schiff base with 15-crown-5 ester, DB-15C5, which was easy to synthesize and highly specific for the sensitive determination of human serum albumins (HSA) in physiological media or human urine. DB-15C5 exhibited weak or no fluorescence in the physiological environment because of the typical twisted intramolecular charge transfer (TICT) properties, but the addition of HSA caused the fluorescence switch-on responses of DB-15C5. DB-15C5 inserted into the α-helices from subdomains IIA (Sudlow site I) and IIIA (Sudlow site II) of HSA. The crown ester core embedded in the central cavity of site I, which involved in π-π stacking interactions and H-bond. The triphenylamine group stretched into the cavity of site II and failed to freely rotate, and the TICT excited state of DB-15C5 was restricted. Good calibration graphs of the response to HSA and a notably low limit of detection (LOD) (1.7nM in PBS, 29.5nM in urine) enabled us to develop a potent functional molecular probe to determine the HSA content in actual biosystems, which was not affected by other proteins and coexisted ions.

Determination of tetrodotoxin in human urine and blood using C18 cartridge column, ultrafiltration and LC–MS

Six fishermen were victims (including one death) of food poisoning from unknown fish on their boat in central Taiwan Strait, in April 2001. The symptoms were like those of tetrodotoxin (TTX) poisoning. As there was no remaining fish, a new protocol was developed to determine TTX in the urine and blood of the victims. The urine and blood samples were cleansed using a C18 Sep-Pak cartridge column, and the toxin was extracted by methanol. The eluate was filtered through a microcentrifuge filter. The filtrate was freeze-dried, dissolved in distilled water, and determined by LC-MS. The recovery was more than 88.9%. The detection limit was 15.6 nM. A linear relationship between response and concentration was obtained between 93.75 and 9375 nM of TTX. It was shown that the urine and blood of the victims contained TTX. The range of TTX was 4.5-40.6 nM in blood and 47-344 nM in urine. Judging from the symptoms of the victims and the experimental data, the causative agent of the food poisoning was identified as TTX.

Enantioselective HPLC method for quantitative determination of tramadol andO-desmethyltramadol in plasma and urine: Application to clinical studies

A sensitive, enantioselective high-performance liquid chromatographic method has been developed for the separation and individual quantitative determination of (+)-and (-)-tramadol and (+)- and (-)-O-desmethyltramadol (M1) in plasma and urine. Extraction from plasma and urine was performed by solid-phase extraction (SPE) on disposable butyl silica (100 mg) extraction cartridges. Separation of the enantiomers of tramadol and M1 was achieved on a Chiralpak AD column containing amylose tris-(3,5-dimethylphenylcarbamate) as chiral selector. The mobile phase was isohexane-ethanol-diethylamine, 97:2.8:0.1 (v/v). Quinidine was used as internal standard. The analytes were detected by use of fluorescence detection. The limit of quantification for tramadol and M1 as 5 nM in plasma and 25 nM in urine. Recoveries were approximately 90% for tramadol and M1 in both plasma and urine. Linearity was observed for both enantiomers of tramadol and M1 in both plasma (r2>0.999) and urine (r2>0.997). The intra and inter-day precision (CV) did not exceed 6.0%. The applicability of the method was demonstrated by means of two clinical studies—a pharmacokinetic study in which a healthy volunteer received 150 mg tramadol hydrochloride as a single oral dose and a study in which poor and extensive CYP2D6 metabolizers received 50 mg tramadol hydrochloride as a single oral dose.

Two pyridinium metabolites of haloperidol are present in the brain of patients at post-mortem.

We have shown in patients taking the antipsychotic drug haloperidol (HP) that two pyridinium metabolites (HPP+ and RHPP+) are present in blood and urine in nM concentrations. These metabolites are structurally analogous to MPP+, the neurotoxic metabolite of the well-known parkinsonian-producing protoxin, MPTP. In this study we measured the concentrations of HPP+ and RHPP+ in seven regions of the brain (putamen, substantia nigra, globus pallidus, caudate, hippocampus, cerebellum and occipital cortex) obtained at post-mortem from three patients who were taking HP before death. Blood, urine, and bile from one patient were analysed as well. HPP+ was present in all regions (except for substantia nigra in one patient and globus pallidus in another); the amount/g ranged from 1.6-8.3 pMol but there was no preferential sequestration of the metabolite in dopaminergic regions. Similarly, RHPP+ was present relatively uniformly in all regions; the amount/g ranged from 1.1-7.6 pMol. The concentrations of HPP+ and RHPP+ in one patient were 24 and 13 nM in blood, 660 and 230 nM in urine, and 13.0 and 1.4 microM in bile, respectively. The presence of these pyridinums in brain adds another important piece of information to the case that, at least for HP, metabolite-induced neurotoxicity could contribute to the extrapyramidal side-effects in patients receiving long-term therapy.

Determination of Elsamitrucin (BMY-28090) in plasma and urine by high-performance liquid chromatography with fluorescence detection.

The cytostatic agent Elsamitrucin is a new fermentation product active in a variety of in vivo tumor models of murine and human origin. To determine its pharmacokinetics during the clinical phase I trial, an HPLC procedure was developed and validated. Plasma samples were extracted after addition of the internal standard, i.e. the analog Chartreusin. Urine samples were injected without extraction of the samples. Because of the wide concentration range of Elsamitrucin in the plasma samples two standard curves were used: up to 100 nM and from 100-1000 nM. Recoveries of Elsamitrucin from plasma were 87% and 74% for concentrations lower and higher than 100 nM, respectively. The detection limits were 1 nM in plasma and 7.5 nM in urine at a signal-to-noise ratio of 3. The accuracy ranged from 95-107% for plasma and from 96-104% for urine. The within-day precision was < or = 4.8% and < or = 2.8% in plasma and urine, respectively. The between-day precision was < or = 4.4% and < or = 7.1% in plasma and urine, respectively. The method proved to be sufficiently sensitive, specific and accurate for analysis of clinical samples for pharmacokinetic purposes.

High-performance liquid chromatographic method for the determination of mangiferin, likviritin and dihydroquercetin in rat plasma and urine

The use of reversed-phase high-performance liquid chromatography for the determination of the biologically active plant phenolic compounds mangiferin, likviritin and dihydroquercetin is described. Perchloric acid (35%) was used for deproteinization in the case of mangiferin and likviritin, and acidified methanol for dihydroquercetin. Detection was performed at 254, 275 and 290 nm for mangiferin, likviritin and dihydroquercetin in plasma, and 365, 312 and 290 nm in urine, respectively. The limit of detection was 0.2 μg/ml for plasma and 0.5 μg/ml for urine.

Quantitative Determination of Metadrenaline and Normetadrenaline in Human Urine

1. A method of measuring the concentrations of M and NM in human urine was described. 2. The normal human excretes daily 100_??_300 μg. of M and almost the same amount of NM in the urine. The concentrations of M and NM in the urine from patients with Graves' disease, essential hypertension, primary aldosteronism and Parkinsonism were within the normal ragne. 3. The concentrations of M and NM in the urine from patients with phaeochromocytoma were abnormally high.