Fluorescent Organic Nanoaggregates for Selective Recognition of d-(-)-Ribose in Biological Fluids and Oral Supplements.

Easily synthesizable, fluorescent, organic nanoaggregates have been utilized, for the first time, in the selective recognition of d-(-)-ribose at pH 5.5 in water. In the self-assembled form, the reactive sites of the monomer units can be properly organized to form an effective "recognition cleft" for ribose (limit of detection ≈23 µm), in which binding mainly occurs through a combination of hydrogen-bonding and CH⋅⋅⋅π interactions. The degree of agglomeration shows a profound influence on the extent of ribose sensing. A reduction in the optical response (≈1.8-fold) is observed when ribose is allowed to interact with nanoaggregates of smaller dimensions (a decrease in the hydrodynamic diameter from (≈212.7±10.2) to (≈44.6±3.5) nm). The protocol is also utilized for the estimation of ribose in human urine samples and oral supplements. Low-cost paper strips have also been developed for rapid, on-site detection of ribose without involving any sophisticated instruments or skilled personnel.

Metal Oxide-Based Selective Enrichment Combined with Stable Isotope Labeling-Mass Spectrometry Analysis for Profiling of Ribose Conjugates.

Some modified ribonucleosides in biological fluids have been evaluated as cancer-related metabolites. Detection of endogenous modified ribonucleosides in biological fluids may serve as a noninvasive cancers diagnostic method. However, determination of modified ribonucleosides is still challenging because of their low abundance and serious matrix interferences in biological fluids. Here, we developed a novel strategy for comprehensive profiling of ribose conjugates from biological fluids using metal oxide-based dispersive solid-phase extraction (DSPE) followed with in vitro stable isotope labeling and double neutral loss scan-mass spectrometry analysis (DSPE-SIL-LC-DNLS-MS). Cerium dioxide (CeO2) was used to selectively recognize and capture ribose conjugates from complex biological samples under basic environment. The enriched ribose conjugates were subsequently labeled with a pair of isotope labeling reagents (acetone and acetone-d6). The glucosidic bond of acetone labeled ribose conjugates is readily ruptured, and the generated ribose that carries an isotope tag can be lost as a neutral fragment under collision induced dissociation (CID). Since the light (acetone) and heavy (acetone-d6) labeled compounds have the same chemical structures and can generate different neutral loss fragments (NL 172 and 178 Da), it is therefore highly convenient to profile ribose conjugates by double neutral loss scan mode in mass spectrometry analysis. In this respect, the light and heavy labeled compounds were ionized at the same condition but recorded separately on MS spectra, which can significantly improve the detection specificity and facilitate the identification of ribose conjugates. Using the developed DSPE-SIL-LC-DNLS-MS strategy, we profiled the ribose conjugates in human urine, and 49 ribose conjugates were readily identified, among which 7 ribose conjugates exhibited significant contents change between healthy controls and lymphoma patients. The DSPE-SIL-LC-DNLS-MS strategy combines the selective enrichment, stable isotope labeling, and double neutral loss scan - MS analysis, which therefore can efficiently minimize false positive results, facilitate the relative quantification, and notably increase the numbers of identified ribose conjugates in biological fluids samples. Taken together, this study established a promising strategy for the effective profiling of urinary modified ribonucleosides, and simultaneous evaluation of the contents change of multiple modified ribonucleosides should provide more accurate and conclusive results for the use of urinary modified ribonucleosides as indicators of cancers.

Nontargeted modification-specific metabolomics study based on liquid chromatography-high-resolution mass spectrometry.

Modifications of genes and proteins have been extensively studied in systems biology using comprehensive analytical strategies. Although metabolites are frequently modified, these modifications have not been studied using -omics approaches. Here a general strategy for the nontargeted profiling of modified metabolites, which we call "nontargeted modification-specific metabolomics", is reported. A key aspect of this strategy was the combination of in-source collision-induced dissociation liquid chromatography-mass spectrometry (LC-MS) and global nontargeted LC-MS-based metabolomics. Characteristic neutral loss fragments that are specific for acetylation, sulfation, glucuronidation, glucosidation, or ribose conjugation were reproducibly detected using human urine as a model specimen for method development. The practical application of this method was demonstrated by profiling urine samples from liver cirrhosis patients. Approximately 900 features were identified as modified endogenous metabolites and xenobiotics. Moreover, this strategy supports the identification of compounds not included in traditional metabolomics databases (HMDB, Metlin, and KEGG), which are currently referred to as "unknowns" in metabolomics projects. Nontargeted modification-specific metabolomics opens a new perspective in systems biology.

Elucidation of the mechanism of ribose conjugation in a pyrazole-containing compound in rodent liver.

1. Here we report on the mechanism of ribose conjugation, through NADH as a cofactor, of a pyrazole-containing compound (PT). Incubation of PT in rat liver microsomes supplemented with NADP⁺/H, NAD⁺/H, and ß-nicotinamide mononucleotide (NMN) resulted in complete conjugation to the adenine dinucleotide phosphate conjugate (ADP-C), adenine dinucleotide conjugate (AD-C), and 5-phosphoribose conjugate (Rib-C1), respectively. In hepatocytes, PT predominantly formed three ribose conjugates: Rib-C1, the ribose conjugate (Rib-C2), and the carboxylic acid of Rib-C2 (Rib-C3). 2. Phosphatase inhibitors were added to hepatocyte incubations. AD-C was detected in this reaction, which suggests that one of the major pathways for the formation of the ribose conjugates is through NAD⁺/H. When AD-C was incubated with phosphatase, Rib-C1 and Rib-C2 formed. 3. To understand the in vivo relevance of this metabolic pathway, rats were dosed with PT and Rib-C2 was found in the urine. 4. Structure-activity relationship shows that replacement of the distal thiazole group in the PT to a phenyl group abolishes this conjugation. Three amino acid residues in the active site preferentially interact with the sulfur atom in the thiazole of PT. 5. In summary, PT forms direct AD-C in hepatocytes, which is further hydrolyzed by phosphatase to give ribose conjugates.

Analysis of urinary metabolic signatures of early hepatocellular carcinoma recurrence after surgical removal using gas chromatography-mass spectrometry.

The objective of present study was to offer insights into the metabolic responses of hepatocellular carcinoma (HCC) to surgical resection and the metabolic signatures latent in early HCC recurrence (one year after operation). Urinary metabolic profiling employing gas chromatography time-of-flight mass spectrometry (GC-TOF MS) was utilized to investigate the complex physiopathologic regulations in HCC after operational intervention. It was revealed that an intricate series of metabolic regulations including energy metabolism, amino acid metabolism, nucleoside metabolism, tricarboxylic acid (TCA) cycle, gut floral metabolism, etc., principally leading to the direction of biomass synthesis, could be observed after tumor surgical removal. Moreover, metabolic differences between recurrent and nonrecurrent patients had emerged 7 days after initial operation. The metabolic signatures of HCC recurrence principally comprised notable up-regulations of lactate excretion, succinate production, purine and pyrimidine nucleosides turnover, glycine, serine and threonine metabolism, aromatic amino acid turnover, cysteine and methionine metabolism, and glyoxylate metabolism, similar to metabolic behaviors of HCC burden. Sixteen metabolites were found to be significantly increased in the recurrent patients compared with those in nonrecurrent patients and healthy controls. Five metabolites (ethanolamine, lactic acid, acotinic acid, phenylalanine and ribose) were further defined; they were favorable to the prediction of early recurrence.

Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis.

The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS(3) screening runs and MS(n(n=4-6)) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N(omega)-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N(6)-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N(6)-methyl-N(6)-threonylcarbamoyladenosine, and 2-methylthio-N(6)-threonylcarbamoyladenosine in human urine is verified for the first time.

Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two folds in both gastric cancer and lymphoma; one ribose conjugate increased more than two folds in both esophagus cancer and lymphoma. The developed analytical method provides a good platform to study the modified ribonucleosides in human body fluids.

Profiling of cis-diol-containing nucleosides and ribosylated metabolites by boronate-affinity organic-silica hybrid monolithic capillary liquid chromatography/mass spectrometry.

RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5'-deoxy-5'-methylthioadensine, N(4)-acetylcytidine, 1-ribosyl-N-propionylhistamine and N(2),N(2),7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers.

Liquid-chromatographic measurement of urinary monosaccharides.

We describe "high-performance" liquid chromatography of urinary monosaccharides on a macroporous anion-exchange resin (Hitachi 3013N), with acetonitrile/water as eluent. The separated xylose, arabinose, D-fructose, mannose, galactose, and D-glucose were detected by means of a post-column reduction reaction of tetrazolium blue. Absolute limits of detection were about 10 ng of monosaccharide. Lactose can also be measured.

Dose-dependent pharmacokinetics of 1-(2-deoxy-beta-D- ribofuranosyl)-2,4-difluoro-5-iodobenzene: a potential mimic of 5-iodo-2'-deoxyuridine.

The dose-range pharmacokinetics of l-(2-deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (5-IDFPdR), a C-aryl nucleoside mimic of IUdR, were studied in male Sprague-Dawley rats following single intravenous (i.v.) and oral doses. After i.v. administration, the blood clearance decreased from approximately 32 ml/min/kg at a dose of 15 mg/kg, to approximately 19 ml/min/kg when dosed at 54 mg/kg, and the elimination half-life increased from 8.4 min to 21.5 min, for the respective doses. While the dose-normalized area under the concentration-time curve (AUCnorm) remained practically unchanged (0.132 kg min ml(-1)) upon increasing the i.v. dose from 5 to 15 mg/kg, it increased by about 44% ( approximately 0.19 kg min ml(-1)) when the i.v. dose was increased from 15 to 54 mg/kg. Similarly, there was a dose-dependent increase in AUCnorm with increasing oral doses: AUCnorm increased by 49% as the oral dose increased from 20 to 40 mg/kg, and further by 55% as the oral dose was increased from 40 mg/kg to 54 mg/kg. For the respective oral doses, the elimination half-life increased from 24.5 min to 176 min, while blood clearance was reduced from approximately 37 ml/min/kg to approximately 17 ml/min/kg. The urinary recoveries of unchanged 5-IDFPdR and its glucuronides (as percent of the dose) were somewhat increased at higher doses. This increase was more pronounced following the highest oral dose. The total biliary recovery of 5-IDFPdR (as percent of the dose) was, however, decreased with increasing doses. The overall kinetic profile of 5-IDFPdR based on these data is suggestive of dose-dependent pharmacokinetics. Decreased elimination of 5-IDFPdR with increasing dose, as supported by longer elimination half-lives at higher doses, is one likely mechanism contributing to the dose-dependent behaviour of this compound. Saturable non-renal metabolism might explain the reduced total body clearance of 5-IDFPdR at higher doses, despite the unchanged or increased urinary clearance. For drugs exhibiting nonlinear kinetics, the dosage regimens may need to be carefully designed to avoid potential unpredictable toxicity and/or lack of pharmacological response associated with the disproportional changes in steady state drug concentrations on changing dose. Manifestation in the rat of nonlinear kinetics at doses of 5-IDFPdR, which may be of therapeutic relevance, warrants extended dose-range evaluations of this compound in future preclinical and clinical studies, to establish safe and efficacious dosage regimens.

Biotransformation of the antiviral agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896) and characteristics of a mesoionic ribose metabolite.

The biotransformation of the antiinfluenza agent 1,3,4-thiadiazol-2-ylcyanamide (LY217896, I) was studied. In addition to a urea metabolite (II) formed by transformation of the cyanamide functionality, another highly polar metabolite was found in mouse urine and in BSC-1, MDCK, and other cell culture incubations of [14C]LY217896. Using 13C-labeled LY217896 together with NMR and MS techniques, this highly polar metabolite was identified as a ribose derivative (III), which apparently exists in a mesoionic form (i.e. positive and negative charges within the same ring system). It was also found that this ribose is formed from LY217896 and ribose-1-phosphate in a reaction catalyzed by the enzyme purine nucleoside phosphorylase, but that the reverse reaction (cleavage of the ribose) is not observed under the conditions used. When tested in vitro using the same assay as that used to measure the antiviral activity of LY217896, this ribose and the urea metabolite exhibit essentially no activity. The presence of a ribose has been implicated in the activity of antiviral compounds such as ribavirin and anticancer agents like 2-aminothiadiazole and tiazofurin, which are structurally similar to LY217896. These activities have been postulated to involve either mono- or triphosphorylated forms, or NAD-type analogs. Possible implications of the formation of this mesoionic ribose metabolite for the mechanism of antiviral activity of LY217896 are discussed.