Creatine riboside is a cancer cell-derived metabolite associated with arginine auxotrophy.

The metabolic dependencies of cancer cells have substantial potential to be exploited to improve the diagnosis and treatment of cancer. Creatine riboside (CR) is identified as a urinary metabolite associated with risk and prognosis in lung and liver cancer. However, the source of high CR levels in patients with cancer as well as their implications for the treatment of these aggressive cancers remain unclear. By integrating multiomics data on lung and liver cancer, we have shown that CR is a cancer cell-derived metabolite. Global metabolomics and gene expression analysis of human tumors and matched liquid biopsies, together with functional studies, revealed that dysregulation of the mitochondrial urea cycle and a nucleotide imbalance were associated with high CR levels and indicators of a poor prognosis. This metabolic phenotype was associated with reduced immune infiltration and supported rapid cancer cell proliferation that drove aggressive tumor growth. CRhi cancer cells were auxotrophic for arginine, revealing a metabolic vulnerability that may be exploited therapeutically. This highlights the potential of CR not only as a poor-prognosis biomarker but also as a companion biomarker to inform the administration of arginine-targeted therapies in precision medicine strategies to improve survival for patients with cancer.

Early pregnancy metabolites predict gestational diabetes mellitus: implications for fetal programming.

BACKGROUND: Aberrant fetal programming in gestational diabetes mellitus seems to increase the risk of obesity, type 2 diabetes, and cardiovascular disease. The inability to accurately identify gestational diabetes mellitus in the first trimester of pregnancy has thwarted ascertaining whether early therapeutic interventions reduce the predisposition to these prevalent medical disorders. OBJECTIVE: A metabolomics study was conducted to determine whether advanced analytical methods could identify accurate predictors of gestational diabetes mellitus in early pregnancy. STUDY DESIGN: This nested observational case-control study was composed of 92 gravidas (46 in the gestational diabetes mellitus group and 46 in the control group) in early pregnancy, who were matched by maternal age, body mass index, and gestational age at urine collection. Gestational diabetes mellitus was diagnosed according to community standards. A comprehensive metabolomics platform measured 626 endogenous metabolites in randomly collected urine. Consensus multivariate criteria or the most important by 1 method identified low-molecular weight metabolites independently associated with gestational diabetes mellitus, and a classification tree selected a subset most predictive of gestational diabetes mellitus. RESULTS: Urine for both groups was collected at a mean gestational age of 12 weeks (range, 6-19 weeks' gestation). Consensus multivariate analysis identified 11 metabolites independently linked to gestational diabetes mellitus. Classification tree analysis selected a 7-metabolite subset that predicted gestational diabetes mellitus with an accuracy of 96.7%, independent of maternal age, body mass index, and time of urine collection. CONCLUSION: Validation of this high-accuracy model by a larger study is now needed to support future studies to determine whether therapeutic interventions in the first trimester of pregnancy for gestational diabetes mellitus reduce short- and long-term morbidity.

Alterations of Proximal Tubular Secretion in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND AND OBJECTIVES: In autosomal dominant polycystic kidney disease (ADPKD), the GFR often remains normal despite significant nephron loss. Proximal tubular secretory clearance may be reduced in ADPKD before detectable changes in GFR. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We used targeted mass spectrometry to quantify secretory solutes from blood and urine samples from 31 patients with ADPKD and preserved GFR (mean eGFR =111±11 ml/min per 1.73 m2) and 25 healthy control individuals as well as from 95 patients with ADPKD and reduced GFR (mean eGFR =53±21 ml/min per 1.73 m2) and 92 individuals with non-ADPKD CKD. We used linear regression to compare the fractional excretion of each solute between ADPKD and control groups. Among 112 patients with ADPKD, we used linear regression to determine associations of solute fractional excretion with height-adjusted total kidney volume. RESULTS: After adjusting for demographics, clinical characteristics, and kidney function measures, the fractional excretions of three secretory solutes were lower in patients with ADPKD and preserved GFR compared with healthy individuals: 52% lower cinnamoylglycine excretion (95% confidence interval, 24% to 70%), 53% lower tiglylglycine excretion (95% confidence interval, 23% to 71%), and 91% lower xanthosine excretion (95% confidence interval, 83% to 95%). In addition to lower excretions of tiglylglycine and xanthosine, patients with ADPKD and reduced GFR also demonstrated 37% lower dimethyluric acid excretion (95% confidence interval, 21% to 50%), 58% lower hippurate excretion (95% confidence interval, 48% to 66%), 48% lower isovalerylglycine excretion (95% confidence interval, 37% to 56%), and 31% lower pyridoxic acid excretion (95% confidence interval, 16% to 42%) compared with patients with non-ADPKD CKD and comparable eGFR. Among patients with ADPKD, solute fractional excretions were not associated with differences in kidney volume. CONCLUSIONS: Patients with ADPKD and preserved and reduced GFR demonstrate lower tubular secretory solute excretion compared with healthy controls and patients with non-ADPKD CKD. Our results suggest that tubular secretion is impaired in ADPKD independent of GFR.

Gallic acid functionalized UiO-66 for the recovery of ribosylated metabolites from human urine samples.

Early diagnosis of disease biomarkers has been focused in recent years through Omics sciences. Nucleosides are the biomarkers of cancers including lung cancer, colorectal cancer, thyroid cancer, bladder cancer, cervical cancer and breast cancer. Nucleosides are directly excreted in the urine of diseased states whereas they are decomposed into other forms as modified nucleosides in healthy conditions. A dual affinity probe (gallic acid modified UiO-66) is prepared and reported for the first time in selectively enriching the ribosylated metabolites and modified nucleosides. Material is characterized by SEM, EDX, FTIR and Nitrogen adsorption porosimetry. The enrichment is benefitted from the interaction ability of zirconium towards glycosylated molecules, rich surface chemistry (3 terminal hydroxyl groups) on gallic acid and high surface area (384 m2/g) of 3-dimensional porous structure of metal organic frameworks (MOFs). Material shows selectivity of 1:500, recovery up to 137.1% and binding capacity of 2340.9 µg/g. Forty-three (43) nucleosides are enriched from human urine samples and 12 potential nucleoside biomarkers from colorectal cancer samples are quantified and their concentration is found higher than in the healthy controls.

Modified Nucleosides - Molecular Markers Suitable for Small-volume Cancer?

BACKGROUND: Modified nucleosides (mNS) in urine are shown to be encouraging markers in cancer, mostly in patients presenting with high tumor mass such is breast and lung cancer. To our knowledge, mNS have not been investigated in head and neck squamous cell carcinoma (HNSCC). HNSCC is characterized by early metastasis into locoregional lymph nodes and slow infiltrating growth, but even in the advanced stage exhibits only a relatively low cancer volume. Therefore, reliable distinction between HNSCC and healthy controls by urinary mNS might pose substantial analytical problems and even more as patients with HNSCC mostly have an increased exposure to tobacco smoke and excessive alcohol consumption which affect the renal mNS pattern. MATERIALS AND METHODS: Urinary mNS in samples of 93 therapy-naive patients with HNSCC and 242 healthy controls were quantified by reversed-phase high-performance liquid chromatography. Considering that the circadian rhythm causes diuresis-induced variations in concentration, the mNS-to-creatinine ratio was chosen to compare patients and controls. For sensitivity and specificity in discriminating between patients and controls, the corresponding curve was plotted. Additionally, logistic regression was carried out and a multilayer perceptron neuronal network (NN) was created. RESULTS: Fifteen mNS were detectable in cases and controls; concentrations of 11 were found to be significantly different. The sensitivity and specificity depend on the total volume of the lesion; HNSCC with volume <20 ml was reliably detected, but those with a volume of 20 ml or greater produced amounts of mNS which led to the most accurate detection of HNSCC based on HNSCC-specific mNS patterns. CONCLUSION: Analysis of urinary mNS allows for detection of small-volume HNSCC, with acceptable specificity and sensitivity if the tumor volume exceeds 20 ml.

Identification of Urinary Metabolite Biomarkers of Type 2 Diabetes Nephropathy Using an Untargeted Metabolomic Approach.

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus (DM). To discover early stage biomarkers of DN, untargeted liquid chromatography-mass spectrometry-based metabolomic analysis was performed in urine samples from healthy subjects and patients with micro- or macroalbuminuria due to nondiabetic disease (macro), type 2 DM without microalbuminuria (T2DM), and type 2 DM with microalbuminuria (T2DM+micro). Levels of four metabolites were significantly different among groups, and they were quantified in a larger group of 267 urine samples. Two metabolites were also discovered and validated in targeted metabolic study of amino acids. For diagnosis of nephropathy, N1-methylguanosine had the highest area-under-the-curve (AUC) value of 0.75 when compared to those of valine (0.68), xanthosine (0.67), and 7-methyluric acid (0.69). After combining fasting blood glucose and diastolic blood pressure (DBP) with N1-methylguanosine, the AUC increased to 0.987. To distinguish between T2DM and T2DM+micro conditions, xanthosine and N1-methylguanosine have AUC value of 0.612 and 0.624, respectively. After adjustment of HbA1c and DBP, AUC values of xanthosine and N1-methylguanosine increased to 0.716 and 0.723, respectively, and could be used to predict the development of nephropathy in T2DM patients.

Nanocoating cellulose paper based microextraction combined with nanospray mass spectrometry for rapid and facile quantitation of ribonucleosides in human urine.

A rapid and facile analytical method for quantification of ribonucleosides in human urine was developed by the combination of nanocoating cellulose paper based microextraction and nanoelectrospray ionization-tandem mass spectrometry (nESI-MS/MS). Cellulose paper used for microextraction was modified by nano-precision deposition of uniform ultrathin zirconia gel film using a sol-gel process. Due to the large surface area of the cellulose paper and the strong affinity between zirconia and the cis-diol compounds, the target analytes were selectively extracted from the complex matrix. Thus, the detection sensitivity was greatly improved. Typically, the nanocoating cellulose paper was immersed into the diluted urine for selective extraction of target analytes, then the extracted analytes were subjected to nESI-MS/MS detection. The whole analytical procedure could be completed within 10min. The method was evaluated by the determination of ribonucleosides (adenosine, cytidine, uridine, guanosine) in urine sample. The signal intensities of the ribonuclesides extracted by the nanocoating cellulose paper were greatly enhanced by 136-459-folds compared with the one of the unmodified cellulose paper based microextraction. The limits of detection (LODs) and the limits of quantification (LOQs) of the four ribonucleosides were in the range of 0.0136-1.258µgL-1 and 0.0454-4.194µgL-1, respectively. The recoveries of the target nucleosides from spiked human urine were in the range of 75.64-103.49% with the relative standard deviations (RSDs) less than 9.36%. The results demonstrate the potential of the proposed method for rapid and facile determination of endogenous ribonucleosides in urine sample.

Unique and Novel Urinary Metabolomic Features in Malignant versus Benign Adrenal Neoplasms.

Purpose: Adrenal incidentalomas must be differentiated from adrenocortical cancer (ACC). Currently, size, growth, and imaging characteristics determine the potential for malignancy but are imperfect. The aim was to evaluate whether urinary small molecules (<800 Da) are associated with ACC.Experimental Design: Preoperative fasting urine specimens from patients with ACC (n = 19) and benign adrenal tumors (n = 46) were analyzed by unbiased ultraperformance liquid chromatography/mass spectrometry. Creatinine-normalized features were analyzed by Progenesis, SIMCA, and unpaired t test adjusted by FDR. Features with an AUC >0.8 were identified through fragmentation patterns and database searches. All lead features were assessed in an independent set from patients with ACC (n = 11) and benign adrenal tumors (n = 46) and in a subset of tissue samples from patients with ACC (n = 15) and benign adrenal tumors (n = 15) in the training set.Results: Sixty-nine features were discovered and four known metabolites identified. Urinary creatine riboside was elevated 2.1-fold (P = 0.0001) in patients with ACC. L-tryptophan, Nε,Nε,Nε-trimethyl-L-lysine, and 3-methylhistidine were lower 0.33-fold (P < 0.0001), 0.56-fold (P < 0.0001), and 0.33-fold (P = 0.0003) in patients with ACC, respectively. Combined multivariate analysis of the four biomarkers showed an AUC of 0.89 [sensitivity 94.7% (confidence interval {CI}, 73.9%-99.1%), specificity 82.6% (CI, 68.6%-92.2%), PPV 69.2% (CI, 48.2%-85.6%), and NPV 97.4% (CI, 86.5%-99.6%)] for distinguishing ACC from benign tumors. Of the four, creatine riboside and four unknown features were validated. Creatine riboside, Nε,Nε,Nε-trimethyl-L-lysine, and two unknown features were elevated in ACC tumors.Conclusions: There are unique urinary metabolic features in patients with ACC with some metabolites present in patient tumor samples. Urinary creatine riboside can differentiate benign adrenal neoplasms from ACC. Clin Cancer Res; 23(17); 5302-10. ©2017 AACR.

The true distribution volume and bioavailability of mizoribine in children with chronic kidney disease.

BACKGROUND: Mizoribine (MZR) is used kidney transplant and various kidney diseases. However, few studies reported the association between pharmacokinetics and pharmacodynamics. The Pharmacokinetics Study Group for Pediatric Kidney Disease (PSPKD) used population pharmacokinetics (PPK) analysis and Bayesian analysis to investigate the usefulness of MZR. In this study, the fact that almost all MZR are excreted unchanged in urine was used to calculate its bioavailability (F) and true distribution volume (V d), and analyzed these correlation with age. METHODS: Ishida et al. reported a PPK analysis by the PSPKD. In the present study, 71 samples extracted from their study population of 105 pediatric chronic kidney disease patients aged between 1 and 20 years were investigated. The bioavailability was calculated by measuring total excreted MZR in 24 h urine samples, and this was compared to the oral dosage. The apparent distribution volume (V d/F) obtained from Bayesian analysis was then used to calculate true distribution volume (V d), and the correlation of each parameter with age was investigated. RESULTS: The median dose of MZR per weight was 5.17 mg/kg/day. Median bioavailability was 32.02%. Median V d per weight was 0.46 L/kg. There was a significant, weakly positive correlation between bioavailability and age (p = 0.026). There was also a significant, weakly negative correlation between V d per weight and age (p = 0.003). CONCLUSION: Bioavailability and V d per weight tended to decrease depending on age. The younger patient required larger dose required to obtain the maximum effect from MZR, and this is important for immunosuppressive therapy.

Immobilization of zirconium-glycerolate nanowires on magnetic nanoparticles for extraction of urinary ribonucleosides.

The authors have immobilized nanowires made from zirconium glycerolate (ZrGly) on magnetite (Fe3O4) nanoparticles by applying a solvothermal growth process using metal-glycerolate as a precursor. The structure and the dissolution-recrystallization mechanism of the resulting Fe3O4@ZrGly composite were investigated by attenuated total reflection-FTIR, energy-dispersive X-ray analysis, thermogravimetric analysis and solid-state cross polarization/magic angle spinning 13C NMR spectroscopy. The interaction between the zirconium glycerolate in Fe3O4@ZrGly and cis-diols leads to efficient adsorption of riboncleosides which then can be quantified by HPLC with UV detection. The sorbent was successfully applied to the selective enrichment of adenosine, cytidine, uridine and guanosine from spiked human urine samples. The detection limit of the method is in the range from 1.7 to 19 ng·mL-1 of nucleosides in spiked human urine, with relative standard deviations of lower than 12.4% and recoveries ranging from 90.6 to 113%. Graphical abstract Fe3O4@ZrGly with high selectivity towards ribonucleosides was designed and applied for quantitation of urinary ribonucleosides.

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.

Polyethyleneimine-grafted boronate affinity materials for selective enrichment of cis-diol-containing compounds.

Polyethyleneimine (PEI)-grafted and 3-acrylamidophenylboronic acid (AAPBA)-functionalized SiO2 boronate affinity materials were synthesized for the selective enrichment of cis-diol-containing compounds. Characterization results of scanning electron microscopy, Fourier transform infrared spectroscopy, elemental analysis, zeta potential, and X-ray photoelectron spectroscopy indicated the successful fabrication of SiO2@PEI-AAPBA materials. Chromatographic separation of test mixtures reveals that SiO2@PEI-AAPBA has high selective enrichment ability for cis-diol-containing compounds. The binding pH between SiO2@PEI-AAPBA and catechol was found to be as low as pH 4.5, while that between SiO2@PEI-AAPBA and adenosine was only ~7.5. This difference might be attributed to the strong electrostatic repulsion between the solid phase and analytes at a low pH. Furthermore, a diphasic separation column was fabricated based on boronate affinity chromatography, C18-reversed-phase chromatography and applied in pressurized capillary electrochromatography (pCEC). Results showed that four polar nucleosides could be well captured by the boronate affinity chromatography (BAC) section and separated by reversed phase pCEC. Finally, SiO2@PEI600-AAPBA-based solid-phase extraction technology was applied to the purification of ribonucleosides in real urine samples, and results of UHPLC-MS/MS revealed that the intensities of the extracted ions (a neutral mass loss of m/z 132.04 Da) of the ribonucleosides were significantly enhanced after the enrichment.

A novel method of liquid chromatography-tandem mass spectrometry combined with chemical derivatization for the determination of ribonucleosides in urine.

Ribonucleosides are the end products of RNA metabolism. These metabolites, especially the modified ribonucleosides, have been extensively evaluated as cancer-related biomarkers. However, the determination of urinary ribonucleosides is still a challenge due to their low abundance, high polarity and serious matrix interferences in urine samples. In this study, a derivatization method based on a chemical reaction between ribonucleosides and acetone to form acetonides was developed for the determination of urinary ribonucleosides. The derivative products, acetonides, were detected by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The methodological evaluation was performed by quantifying four nucleosides for linear range, average recovery, precision, accuracy and stability. The validated procedures were applied to screen modified ribonucleosides in urine samples. Improvement of separation and enhancement of sensitivity were obtained in the analysis. To identify ribonucleosides, inexpensive isotope labeling acetone (acetone-d6) and label-free acetone were applied to form ordinary and deuterated acetonides, respectively. The two groups of samples were separated with orthogonal partial least squares (OPLS). The ordinary and deuterated pairs of acetonides were symmetrically distributed in the S-plot for easy and visual signal identification. After structural confirmation, a total of 56 ribonucleosides were detected, 52 of which were modified ribonucleosides. The application of derivatization, deuterium-labeling and multivariate statistical analysis offers a new option for selective detection of ribonucleosides in biological samples.

Facile preparation of biocompatible sulfhydryl cotton fiber-based sorbents by "thiol-ene" click chemistry for biological analysis.

Sulfhydryl cotton fiber (SCF) has been widely used as adsorbent for a variety of metal ions since 1971. Thanks to the abundant thiols on SCF, in this study, we reported a universal method for the facile preparation of SCF-based materials using "thiol-ene" click chemistry for the first time. With the proposed method, two types of SCF-based materials, phenylboronic acid grafted sulfhydryl cotton fiber (SCF-PBA) and zirconium phosphonate-modified sulfhydryl cotton fiber (SCF-pVPA-Zr(4+)), were successfully prepared. The grafted functional groups onto the thiol group of SCF were demonstrated by X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDX). The prepared fibrous materials exhibited excellent fiber strength, good stability in aqueous or nonaqueous solutions, and great biocompatibility. Moreover, we developed filter-free in-pipet-tip SPE using these SCF-based materials as adsorbent for the enrichment of ribonucleosides, glycopeptides and phosphopeptides. Our results showed that SCF-PBA adsorbent can selectively capture ribonucleosides and glycopeptides from complex biological samples. And SCF-pVPA-Zr(4+) adsorbent exhibited high selectivity and capacity in the enrichment of phosphopeptides from the digestion mixture of ß-casein and bovine serum albumin (BSA), as well as human serum and nonfat milk digest. Generally, the preparation strategy can be a universal method for the synthesis of other functionalized cotton-based adsorbents with special requirement in microscale biological analysis.

Metabolomic analysis reveals a unique urinary pattern in normozoospermic infertile men.

Normozoospermic infertility has become a common and important health problem worldwide. We designed this metabolomic case-control study to investigate the possible mechanism and urinary biomarkers of normozoospermic infertility. Normozoospermic infertile cases (n = 71) and fertile controls (n = 47) were recruited. A urinary metabolome pattern could discriminate normozoospermic infertile cases from fertile controls. A total of 37 potential biomarkers were identified; these have functionally important roles in energy production, antioxidation, and hormone regulation in spermatogenesis. This gave rise to a combined biomarker pattern of leukotriene E4, 3-hydroxypalmitoylcarnitine, aspartate, xanthosine, and methoxytryptophan pointing to a diagnostic capability (AUC = 0.901, sensitivity = 85.7%, and specificity = 86.8%) in a ROC model; these markers may highlight keynote events of normozoospermic infertility. Stalled medium- and long-chain fatty acid metabolism with improved ketone body metabolism, plus decreased levels of malate and aspartate could result in citrate cycle alterations via a malate-aspartate shuttle in ATP generation in spermatogenesis. Inhibitory alterations in the normal hormone-secreting activity in spermatogenesis were suggested in normozoospermic infertility. Folate deficiency and oxidative stress may jointly impact infertile patients. The disruption of eicosanoid metabolism and xanthine oxidase system, which were tightly associated with energy metabolism and oxidative stress, was also a potential underlying mechanism. In addition, depression might be associated with normozoospermic infertility via neural activity-related metabolites. This study suggests that the urinary metabolome can be used to differentiate normozoospermic infertile men from fertile individuals. Potential metabolic biomarkers derived from these analyses might be used to diagnose what remains a somewhat idiopathic condition and provide functional insights into its pathogenesis.

Noninvasive urinary metabolomic profiling identifies diagnostic and prognostic markers in lung cancer.

Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1,500 Da) were measured in urine collected from 469 patients with lung cancer and 536 population controls using unbiased liquid chromatography/mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further confirmed in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.

The need for vigilance: false-negative screening for adenylosuccinate lyase deficiency caused by deribosylation of urinary biomarkers.

OBJECTIVES: Adenylosuccinate lyase deficiency (dADSL) is a rare inherited metabolic disorder. Biochemical diagnosis of the disease is based on the determination of enormously elevated urinary levels of succinylaminoimidazole carboxamide riboside (SAICA-riboside) and succinyladenosine (SAdo). We report a case of false negative screening for dADSL caused by deribosylation of the urinary biomarkers SAICA-riboside and SAdo. DESIGN AND METHODS: A thin-layer chromatography (TLC) method with Pauly reagent detection of SAICA-riboside was used as a screening method. High-performance liquid chromatography with diode-array detection (HPLC-DAD) and LC-MS/MS methods were used for the identification and quantitative determination of SAICA-riboside, SAdo, succinylaminoimidazole carboxamide (SAICA) and succinyladenine (SA). RESULTS: Following a negative TLC screening in a known case of dADSL, we analyzed urine using HPLC-DAD. The concentration of SAICA-riboside was 2.7mmol/mol creatinine (below the TLC detection limit), and we detected the two abnormal metabolites identified by LC-MS/MS as SAICA and SA. We showed that SAICA and SA were produced by deribosylation of SAICA-riboside and SAdo in the patient's urine. Studies performed by monitoring the production of SAICA and SA after the addition of SAICA-riboside and SAdo to the patient's urine and to urine samples from patients with urinary tract infections suggested that deribosylation is facilitated by bacterial enzymes. CONCLUSIONS: Screening methods for the diagnosis of dADSL may be falsely negative due to bacteria-mediated deribosylation of SAICA-riboside and SAdo. HPLC-DAD or LC-MS/MS analyses allowing for simultaneous detection of SAICA-riboside, SAdo and their deribosylation products SAICA and SA should be preferentially used for the diagnosis of dADSL in urine.

Metabolite target analysis of human urine combined with pattern recognition techniques for the study of symptomatic gout.

Recurrent attacks and irregularity are two important characteristics of gout disease. Uric acid as a single evaluation indicator for clinical diagnosis is insufficient considering the versatile properties of gout. The aim of this work is to identify several endogenous metabolites from urine samples for the elucidation and prediction of gout disease. Metabolite target analysis was established for human urine by high performance liquid chromatography-diode array detection (HPLC-DAD). The targeted metabolites selected included hippuric acid, uracil, phenylalanine, tryptophan, uric acid and creatinine as well as nine purine compounds. Useful information was extracted from multivariate data through Fisher Linear Discriminant Analysis (FDA) and Orthogonal Signal Correction Partial Least Squares Discriminant Analysis (OSC-PLS-DA). Uric acid, hypoxanthine, xanthosine, guanosine, inosine and tryptophan were identified as important metabolites among the acute and chronic gout and controls. Based on OSC-PLS-DA models, the regression equations obtained could discriminate gout from the controls as well as the acute from chronic. The recognition and prediction ability is respectively 100% and 85.0% for the gout, 100% and 83.3% for the acute, and 90.91% and 89.9% for the chronic. Metabolic dysfunction of tryptophan and excessive metabolism of xanthosine and hypoxanthine to xanthine were confirmed for gout disease. Metabolic dysfunction of tryptophan was also proven to be induced by allopurinol in case of Kunming mice with hyperuricemia. Potential biomarkers can be used not only to distinguish gout patients from healthy people, but also to evaluate the disease state.

Effect of genetic polymorphisms of SLC28A1, ABCG2, and ABCC4 on bioavailability of mizoribine in healthy Japanese males.

The aim of the present study was to investigate the genetic factors responsible for the interindividual variability in the bioavailability of mizoribine. Thirty healthy Japanese men aged 20-49 years and weighing 53-75 kg participated in the present study and took 150 mg of mizoribine. Urine samples were collected periodically for 12 h after the dose, and the bioavailability of mizoribine was calculated from the estimated total urinary excretion from time zero to infinity. The bioavailability of mizoribine in the 30 subjects ranged from 60.3% to 99.4%. The mean bioavailability of mizoribine in subjects with the concentrative nucleoside transporter 1 (SLC28A1) 565-A/A allele (75.4%) was significantly lower than that in subjects with the SLC28A1 565-G/G allele (90.1%). On the other hand, the bioavailability of mizoribine was not affected by polymorphisms of breast cancer resistance protein (ABCG2) C421A and multidrug resistance-associated protein 4 (ABCC4) G2269A. The findings in the present prospective study suggested that the genetic test for the SLC28A1 G565A polymorphism is promising for predicting the Japanese subjects with lower bioavailability of mizoribine.

Evaluation of urinary ribonucleoside profiling for clinical biomarker discovery using constant neutral loss scanning liquid chromatography/tandem mass spectrometry.

The patterns and levels of urinary excreted ribonucleosides which reflect RNA turnover and metabolism in humans offer the potential for early detection of disease and monitoring of therapeutic intervention. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method employing constant neutral loss (CNL) scanning for the loss of the ribose moiety (132 u) was used to detect ribonucleosides in human urine and to evaluate this analytical platform for biomarker research in clinical trials. Ribonucleosides were stable and not influenced by the time spent at room temperature prior to freezing or long-term storage at -80 °C. Matrix effects caused variation in the mass spectrometer response which was dependent on the concentration of the analysed urine sample. For the use of urinary ribonucleoside profiling in clinical biomarker studies, adjustment of the urine samples to a common concentration prior to sample preparation is therefore advocated. Changes in the mass spectrometer response should be accounted for by the use of an internal standard added after sample preparation. Diurnal variation exceeded inter-day variation of an individual's ribonucleoside profile, but inter-person differences were predominant and allowed the separation of individuals against each other in a multivariate space. Due to considerable diurnal variation the use of spot urine samples would introduce unnecessary variation and should be replaced by the collection of multiple spot urine samples across the day, where possible. Should such a protocol not be feasible, biological intra-day and inter-day variation must be considered and accounted for in the data interpretation.