Biomarkers In Urine Samples Research Articles

Abstract A006: Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms

Abstract Introduction and Objectives Oncuria® is a bead-based multiplex fluorescence immunoassay that coordinately measures 10 protein biomarkers in urine samples. The current study compared assay performance and output when urine samples were evaluated with the Oncuria assay using three different fluorescence-analyzing instruments commonly used in diagnostic laboratories worldwide. Methods We compared the performance of the clinically validated Oncuria bladder cancer (BC) multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96 well microtiter plates, and consecutively evaluated on the LED/image based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX). The BC assay uses magnetic bead based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically <5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusions In conclusion, the Oncuria BC assay performed similarly well across three different flow analysis platforms for all 10 analytes simultaneously evaluated in urine samples. This agreement across instruments indicates that the test is amenable to standardized performance in laboratories using existing xMAP, without requiring costly outlays for new equipment. Citation Format: Toru Sakatani, Sunao Tanaka, Kaoru Murakami, Richard Waldron, Wayne Hogrefe, Charles Rosser, Hideki Furuya. Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A006.

An Eco-Friendly alternative for voltammetric determination of creatinine in urine sample using copper(II) immobilized on biochar

Creatinine (CRE) is the final product of creatine and phosphocreatine metabolism in mammals. CRE is primarily excreted by the kidneys, and a decrease in urinary creatinine content can be an indicator of kidney failure. Therefore, monitoring this biomarker in urine samples is crucial. For this purpose, an electrochemical sensor had been proposed for indirect determination of the target based on the immobilization of copper ions on biochar, an eco-friendly material. First, copper ions (Cu2+) were spontaneously preconcentrated at the surface of a biochar-modified carbon paste electrode. Next, CRE was spontaneously accumulated over this surface, resulting in the suppression of the copper voltammetric signal. This suppression was attributed to the formation of a creatinine-copper complex, and used as the analytical response. A linear dynamic range (LDR) of 300–700 µmol L−1 in the presence of a synthetic urine matrix was achieved, along with limits of detection (LOD) and quantification (LOQ) of 91.0 µmol L−1 and 300 µmol L−1, respectively. Selectivity was suitable for the most common metallic species found in urine and equimolar uric acid concentration. The standard addition method was adopted for CRE determinations in synthetic urine samples, and the recoveries values were 101 ± 7 % for the method's limit of quantification (LOQ) concentration (300 µmol L−1), and 100 ± 7 % for the usual creatinine concentration in urine after a dilution step, 500 µmol L−1. Therefore, an approach based on metal immobilization, using an eco-friendly material, was proposed for indirect creatinine determination, and successfully applied to biomarker analysis in a synthetic urine matrix.

Evaluation of metabolomics-based urinary biomarker models for recognizing major depression disorder and bipolar disorder

BackgroundMajor depressive disorder (MDD) and bipolar disorder (BD) are psychiatric disorders with overlapping symptoms, leading to high rates of misdiagnosis due to the lack of biomarkers for differentiation. This study aimed to identify metabolic biomarkers in urine samples for diagnosing MDD and BD, as well as to establish unbiased differential diagnostic models. MethodsWe utilized a metabolomics approach employing ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) to analyze the metabolic profiles of urine samples from individuals with MDD (n = 50), BD (n = 12), and healthy controls (n = 50). The identification of urine metabolites was verified using MS data analysis tools and online metabolite databases. ResultsTwo diagnostic panels consisting of a combination of metabolites and clinical indicators were identified—one for MDD and another for BD. The discriminative capacity of these panels was assessed using the area under the receiver operating characteristic (ROC) curve, yielding an area under the curve (AUC) of 0.9084 for MDD and an AUC value of 0.9017 for BD. ConclusionsHigh-resolution mass spectrometry-based assays show promise in identifying urinary biomarkers for depressive disorders. The combination of urine metabolites and clinical indicators is effective in differentiating healthy controls from individuals with MDD and BD. The metabolic pathway indicating oxidative stress is seen to significantly contribute to depressive disorders.

Abstract 3773: Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms

Abstract Introduction and Objectives: Oncuria® is a bead-based multiplex fluorescence immunoassay that coordinately measures 10 protein biomarkers in urine samples. The current study compared assay performance and output when urine samples were evaluated with the Oncuria assay using three different fluorescence-analyzing instruments commonly used in diagnostic laboratories worldwide. Methods: We compared the performance of the clinically validated Oncuria bladder cancer (BC) multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96 well microtiter plates, and consecutively evaluated on the LED/image based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX). The BC assay uses magnetic bead based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. Results: All three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically <5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample. Conclusions: In conclusion, the Oncuria BC assay performed similarly well across three different flow analysis platforms for all 10 analytes simultaneously evaluated in urine samples. This agreement across instruments indicates that the test is amenable to standardized performance in laboratories using existing xMAP, without requiring costly outlays for new equipment. Citation Format: Sunao Tanaka, Toru Sakatani, Kaoru Murakami, Richard T. Waldron, Walter Morales, Wayne Hogrefe, Charles J. Rosser, Hideki Furuya. Bladder cancer risk stratification with the Oncuria 10 plex bead-based urinalysis assay using three different Luminex xMAP instrumentation platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3773.

Comparison of Biomarkers of Exposure in a Controlled Study of Smokers Switched from Conventional Cigarettes to Heated Tobacco Products.

The heated tobacco product (HTP) heats rather than burns tobacco to release an aerosol with significantly fewer toxicants than conventional cigarette smoke and has received global attention in recent years. To investigate whether changes in biomarkers of exposure could be detected after switching from conventional cigarettes (CCs) to HTPs, 224 subjects from four cities in China participated in this study. Nine biomarkers containing tobacco-specific nitrosamines (TSNAs), volatile organic compounds (VOCs), and the biomarkers for acrolein and crotonaldehyde were determined by UPLC-MS/MS. The levels of the sum of nine biomarkers in CCs were 5.4 and 5.2 times higher than in an Original-HTP and Menthol-HTP, respectively. Among the nine biomarkers, 3HPMA and 3HMPMA accounted for the highest proportions. Switching from CCs to HTPs is good for both men and women because the changes in each biomarker in urine samples were the same in men and women. Among all the subjects, subjects aged 20-39 years had the greatest reduction in biomarker residues in urine. The findings of the present study provided useful information for the health risk research of HTPs in China.

Deep eutectic solvent-based dispersive liquid-liquid microextraction coupled with HPLC and central composite design for biological monitoring of hippuric acid and methyl hippuric acid in urine

Exposure assessment of toluene and xylene as two hazardous compounds in occupational settings are great of importance. Hippuric acid (HA) and methyl hippuric acid (MHA) are common urinary metabolites for biological monitoring of toluene and xylene exposure, respectively. In this study a dispersive liquid–liquid microextraction (DLLME) method based on a deep eutectic solvent (DES), was suggested to determine the urinary MHA and HA simultaneously. Optimizations of all parameters affecting the DLLME-DES were done using response surface methodology. In obtained optimized conditions, a linear calibration range of 0.024–20 µg mL−1 for HA and 0.033–20 µg mL−1 for MHA were achieved and R2 was above 0.99 for these two metabolites. The detection limit of HA and MHA was 0.008 and 0.010 µg mL−1 respectively. The quantification limit was reported as 0.024 and 0.033 µg mL−1 for HA and MHA respectively. The suggested analytical technique was used for quantitation of target biomarkers in urine samples of occupational workers with high accuracy (97–103.6% for HA and 92–104% MHA) and precision (RSD ≤ 4.5% for HA (RSD ≤ 6.2% for MHA). Applying the proposed method for urine sample analysis of exposed-workers and comparing with the standard method (National Institute for Occupational Safety & Health standard method) showed that HA and MHA can be pre-concentrated and quantified by a simple, fast and environmental friendly method.

Application of Parallel-DPX for the determination of potential cancer biomarkers in urine samples using cork as natural extraction phase followed by GC–MS

Early-stage diagnosis is important to discover possible cancers in the human body. Bioanalytical methods are tools that allow for these studies. Biogenic amines are markers of several cancers in the organism, such as colorectal, lung and pancreatic. In this study, the determination of three important amines, putrescine (Put), spermidine (Spd) and cadaverine (Cad), was proposed. The use of Parallel-DPX-Cork is demonstrated for the first time in this type of application, permitting an effective methodology based on Green Analytical Chemistry (GAC) principles, with separation/quantification by GC–MS in human urine. Multivariate and univariate designs were applied, and the sample was prepared as follows: 10 min for alkaline hydrolysis under agitation with NaOH (2 mol L−1), added in a proportion of 1:8 (v/v) with the sample, at 60 °C, followed by 10 min of centrifugation at 3500 rpm, 5 min for derivatization with chloroformate isobutyl (180 µL), sample pH adjusted at 12; the extraction step was performed using 6 cycles, with 2 mL for each cycle (totalizing 12 mL of sample per experiment) and 25 mg of cork; the desorption step was carried out using 2 cycles with ethyl acetate (300 µL). LODs and LOQs were 15.2 and 50 ng mL−1 for all analytes. Intraday and interday precisions ranged from 2 to 22% and 9 to 28%, respectively. For the relative recoveries, the range was 62% to 130%. Five urine samples, collected from male and female volunteers, were analyzed using the methodology proposed, and the analytes concentrations obtained lower than LOQ and LOD.

Evaluation of NAG, NGAL, and KIM-1 as Prognostic Markers of the Initial Evolution of Kidney Transplantation.

Kidney transplantation is the best option for end-stage chronic kidney disease. Transplant viability is conditioned by drugs' nephrotoxicity, ischemia-reperfusion damage, or acute rejection. An approach to improve graft survival is the identification of post-transplant renal function prognostic biomarkers. Our objective was to study three early kidney damage biomarkers (N-acetyl-d-glucosaminidase, NAG; neutrophil gelatinase-associated lipocalin, NGAL; and kidney injury molecule-1, KIM-1) in the initial period after transplantation and to identify possible correlations with main complications. We analysed those biomarkers in urine samples from 70 kidney transplant patients. Samples were taken on days 1, 3, 5, and 7 after intervention, as well as on the day that renal function stabilised (based on serum creatinine). During the first week after transplant, renal function improved based on serum creatinine evolution. However, increasing levels of biomarkers at different times during that first week could indicate tubular damage or other renal pathology. A relationship was found between NGAL values in the first week after transplantation and delayed graft function. In addition, higher NAG and NGAL, and lower KIM-1 values predicted a longer renal function stabilisation time. Therefore, urinary NAG, NGAL, and KIM-1 could constitute a predictive tool for kidney transplant complications, contributing to improve graft survival rates.

A cross sectional pilot study to assess the role of phthalates on respiratory morbidity among patients with chronic obstructive pulmonary disease

BackgroundChronic Obstructive Pulmonary Disease (COPD) affects ∼16 million U.S. adults. Phthalates, synthetic chemicals in consumer products, may adversely impact pulmonary function and airway inflammation; however, their role on COPD morbidity remains unknown. ObjectiveWe examined associations between phthalate exposures and respiratory morbidity among 40 COPD patients who were former smokers. MethodsWe quantified 11 phthalate biomarkers in urine samples collected at baseline in a 9-month prospective cohort study in Baltimore, Maryland. COPD baseline morbidity measures included: health status and quality of life measures (CAT: COPD Assessment Test, CCQ: Clinical COPD Questionnaire, SGRQ: St. George's Respiratory Questionnaire; mMRC: Modified Medical Research Council Dyspnea Scale), and lung function. Information on prospective exacerbation data was monitored monthly during the 9-month longitudinal follow-up period. To examine associations between morbidity measures and phthalate exposures, we used multivariable linear and Poisson regression models for continuous and count outcomes, respectively, adjusting for age, sex, race/ethnicity, education, and smoking pack-years. ResultsHigher mono-n-butyl phthalate (MBP) concentrations were associated with increased CAT(β, 2.41; 95%CI, 0.31–4.51), mMRC (β, 0.33; 95%CI 0.11–0.55), and SGRQ (β, 7.43; 95%CI 2.70–12.2) scores at baseline. Monobenzyl phthalate (MBzP) was also positively associated with CCQ and SGRQ scores at baseline. Higher concentrations of the molar sum of Di (2-ethylhexyl) phthalate (DEHP) were associated with increased incidence of exacerbations during the follow-up period (incidence rate ratio, IRR = 1.73; 95%CI 1.11, 2.70 and IRR = 1.94; 95%CI 1.22, 3.07, for moderate and severe exacerbations, respectively). MEP concentrations were inversely associated with incidence of exacerbations during the follow-up period. ConclusionsWe found that exposure to select phthalates was associated with respiratory morbidity among COPD patients. Findings warrant further examination in larger studies given widespread phthalate exposures and potential implications for COPD patients should relationships observed be causal.

Evaluation of the Roche point of care system for determination of NT-proBNP in urine samples

IntroductionThe diagnostic potential of urine NT-pro-BNP has been studied recently and shows great promise, especially for children. Bedside serum-NT-proBNP determination with point of care testing (POCT) is established in adults. This study investigated the analytical capabilities of NT-proBNP POCT measurements for urine-samples. Furthermore, the stability of this biomarker in urine samples at room-temperature was explored. Methods104 urine samples were analysed, partly with double and triple determinations and compared to the established Elecsys system. To investigate the stability of NT-proBNP in urine, five samples were stored at room temperature and analysed 12-hourly over 96 h. Results34 % of samples lay outside the POCT range (60 pg/ml-9000 pg/ml). The 69 values within the range were correlated with those measured by Elecsys (correlation coefficient of 0.92; p < 0.001). Linear regression yields an r2 of 0.84. The stability of urine NT-proBNP was shown with correlation coefficients between 0.97 and 1 (p < 0.001). ConclusionAnalysing NT-ProBNP in urine with cobas h232 POC system is feasible, but the detection range needs to be adjusted. As urine NT-proBNP proved stable for at least 96 h even postal shipping would be possible. Adjusting this system‘s measuring range could help introduce a new, non-invasive, pain-free parameter.

P439 Biomarker detection for the diagnosis of disseminated histoplasmosis in people living with HIV/ AIDS in Southern Brazil: a year of implementation

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMBackground: Disseminate histoplasmosis (DH) is an important opportunistic disease in HIV/AIDS patients.Objective: We aimed to report partial results of the implementation of the Histoplasma urinary biomarker as a routine test for the diagnosis of DH in people living with HIV/AIDS (PLWHA) admitted to a reference hospital in Southern Brazil.MethodsThe study was performed in a tertiary hospital, which is recognized as a regional reference center to 21 cities for the treatment of PLWHA. At this service, since the beginning of 2021, a new protocol for DH investigation was adopted, including the systematic execution of urinary antigen test for histoplasmosis (Histoplasma GM EIA, IMMY®) in PLWHA. We retrospectively evaluated data from all of these patients diagnosed with DH in the last 1 year, between March 2021 and March 2022. The study was approved by our university ethics committee (CEP/FURG; N° 234/2018).ResultsWithin the 12 months period, 243 patients from our hospital were investigated by Histoplasma GM EIA through urine samples. A total of 19 were diagnosed with DH through this test, resulting in a prevalence of 7.8%. Interestingly, nine of them were also investigated by antibody detection through immunodiffusion test (IMMY), being all negative in this serology. DH was the AIDS-defining illness in 21% of these patients (4/19), and co-infection with Mycobacterium tuberculosis was detected in 37% (7/19). Half of the patients (11/19) were severe immunosuppressed (CD4 + lymphocytes <100 cells/mm3), 21% (n = 4) had rates between 100 and 200 cells/mm3 and 21% (n = 4) had >200 cells/mm3. The majority of the patients (74%; n = 14) had advanced signs of DH, showing pulmonary impairment, weight loss, fever, neurological, and/or skin lesions and lymphadenopathy, suggesting late diagnosis, 16% (3/19) of the patients had few signs of DH (suggesting precocity), and two patients were diagnosed before the onset of the clinical manifestation, with the strategy of the use of GM Histoplasma EIA as a screening test. Although the antifungal treatment, five of them (35.7%) died.ConclusionThe systematic use of the test to detect a specific biomarker in urine samples had improved substantially the diagnosis of DH in PLWHA in our hospital (19 cases in one year). In addition, since we could diagnose DH with precocity in five patients, we suggest that in regions where DH is hyperendemic, the GM Histoplasma EIA could be used as a screening test for all AIDS patients, similar to what is preconized to cryptococcal disease. More robust studies are instigated to evaluate this strategy, and its benefits to an early diagnosis, resulting in a better quality of life, better prognosis, and consequently reduction of the mortality rate.

Discovery of metabolomic biomarkers for discriminating platinum-sensitive and platinum-resistant ovarian cancer by using GC-MS.

This study aims to determine ovarian cancer (OC) patients with platinum resistance for alternative treatment protocols by using metabolomic methodologies. Urine and serum samples of platinum-resistant and platinum-sensitive OC were analyzed using GC-MS. After data processing of GC-MS raw data, multivariate analyses were performed to interpret complex data for biologically meaningful information and to identify the biomarkers that cause differences between two groups. The biomarkers were verified after univariate, multivariate, and ROC analysis. Finally, metabolomic pathways related to group separations were specified. The results of biomarker analysis showed that 3,4-dihydroxyphenylacetic acid, 4-hydroxybutyric acid, L-threonine, D- mannose, and sorbitol metabolites were potential biomarkers in urine samples. In serum samples, L-arginine, linoleic acid, L-glutamine, and hypoxanthine were identified as important biomarkers. R2Y, Q2, AUC, sensitivity and specificity values of platinum-resistant and sensitive OC patients' urine and serum samples were 0.85, 0.545, 0.844, 91.30%, 81.08 and 0.570, 0.206, 0.743, 77.78%, 74.28%, respectively. In metabolic pathway analysis of urine samples, tyrosine metabolism and fructose and mannose metabolism were found to be statistically significant (p < 0.05) for the discrimination of the two groups. While 3,4-dihydroxyphenylacetic acid, L-tyrosine, and fumaric acid metabolites were effective in tyrosine metabolism. D-sorbitol and D-mannose metabolites were significantly important in fructose and mannose metabolism. However, seven metabolomic pathways were significant (p < 0.05) in serum samples. In terms of p-value, L-glutamine in the nitrogen metabolic pathway from the first three pathways; L-glutamine and pyroglutamic acid metabolites in D-glutamine and D-glutamate metabolism. In the arginine and proline metabolic pathway, L-arginine, L-proline, and L-ornithine metabolites differed significantly between the two groups.

Microchip Immunoassays for Monitoring Renal Function: Rapid, Low-Cost, and Highly Sensitive Quantification of Urinary Biomarkers of Diabetic Nephropathy.

This study developed low-cost and highly sensitive immunoassay devices possessing the ability to rapidly analyze urine samples. Further, they can quantitatively detect three biomarkers indicating renal injury: monocyte chemotactic protein 1 (MCP-1), angiotensinogen (AGT), and liver-type fatty acid binding protein (L-FABP). The devices were used to successfully estimate the concentrations of the three biomarkers in urine samples within 2 min; the results were consistent with those obtained via conventional enzyme-linked immunosorbent assay (ELISA), which requires several hours. In addition, the estimated detection limits for the three biomarkers were comparable to those of commercially available ELISA kits. Thus, the proposed and fabricated devices facilitate high-precision and frequent monitoring of renal function.

Assessment of Human Exposure to Deoxynivalenol, Ochratoxin A, Zearalenone and Their Metabolites Biomarker in Urine Samples Using LC-ESI-qTOF.

Human are exposed to a wide range of mycotoxins through dietary food intake, including processed food. Even most of the mycotoxin exposure assessment studies are based on analysis of foodstuffs, and evaluation of dietary intake through food consumption patterns and human biomonitoring methods are rising as a reliable alternative to approach the individual exposures, overcoming the limitations of the indirect dietary assessment. In this study, human urine samples were analyzed, seeking the presence of deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZEA), and their metabolites. For this purpose, 40 urine samples from female and male adult residents in the city of Valencia (Spain) were evaluated by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF) after salting-out liquid–liquid extraction. Analytical data showed that 72.5% of analyzed samples were contaminated by at least one mycotoxin at variable levels. The most prevalent mycotoxins were de-epoxy DON (DOM-1) (53%), ZEA (40%), and α-zearalenol (αZOL) (43%), while OTA was only detected in one sample. The mean concentrations in positive samples were DON (9.07 ng/mL), DOM-1 (20.28 ng/mL), ZEA (6.70 ng/mL), ZEA-14 glucoside (ZEA-14-Glc) (12.43 ng/mL), αZOL (27.44 ng/mL), αZOL-14 glucoside (αZOL-14-Glc) (12.84 ng/mL), and OTA (11.73 ng/mL). Finally, probable daily intakes (PDIs) were calculated and compared with the established tolerable daily intakes (TDIs) to estimate the potential risk of exposure to the studied mycotoxins. The calculated PDI was below the TDI value established for DON in both female and male adults, reaching a percentage up to 30%; however, this percentage increased up to 92% considering total DON (DON + DOM-1). On the other hand, the PDI obtained for ZEA and its metabolites were higher than the TDI value fixed, but the low urine excretion rate (10%) considered should be highlighted. Finally, the PDI calculated in the detected positive sample for OTA exceeded the TDI value. The findings of the present study confirm the presence of the studied mycotoxins and their metabolites as some of the most prevalent in urine.

Identification of novel neuroblastoma biomarkers in urine samples

Urine is a complex liquid containing numerous small molecular metabolites. The ability to non-invasively test for cancer biomarkers in urine is especially beneficial for screening child patients. This study attempted to identify neuroblastoma biomarkers by comprehensively analysing urinary metabolite samples from children. A total of 87 urine samples were collected from 54 participants (15 children with neuroblastoma and 39 without cancer) and used to perform a comprehensive analysis. Urine metabolites were extracted using liquid chromatography/mass spectrometry and analysed by Metabolon, Inc. Biomarker candidates were extracted using the Wilcoxon rank sum test, random forest method (RF), and orthogonal partial least squares discriminant analysis (OPLS-DA). RF identified three important metabolic pathways in 15 samples from children with neuroblastoma. One metabolite was selected from each of the three identified pathways and combined to create a biomarker candidate (3-MTS, CTN, and COR) that represented each of the three pathways; using this candidate, all 15 cases were accurately distinguishable from the control group. Two cases in which known biomarkers were negative tested positive using this new biomarker. Furthermore, the predictive value did not decrease in cases with a low therapeutic effect. This approach could be effectively applied to identify biomarkers for other cancer types.

Occupational exposure evaluation of Brazil university community to the volatile organic compounds

Occupational exposure to volatile organic compounds (VOC) might generate serious worker health damages. Therefore, biological monitoring is essential to evaluate exposure biomarkers from highly toxic chemicals, ensuring better attention to the worker health. In this study was developed and validated a bioanalytical method based on high-performance liquid chromatography coupled to photodiode array (HPLC-PDA) for the quantification of VOC biomarkers in urine samples from Federal University of Goias (UFG) workers. Samples were collected from 30 occupationally exposed subjects after application of a questionnaire survey. The following biomarkers hippuric acid, methyl-hippuric acid, mandelic acid, phenylglyoxylic acid and phenol were quantified, representing exposition to toluene, xylene, styrene, ethylbenzene, benzene and phenol solvents, respectively. Hippuric acid levels were found close to or above the reference values, although a subject had levels higher than preconized by Biological Limit Values (BLV) guideline of 4.0 mg/g creatinine. Five subjects had 3 and 4-methylhippuric acid ranging from 0.1 to 1.0 mg/g creatinine. These results indicate a moderate to high VOC exposure from UFG workers. Multivariate analysis generated four clusters and indicated that histotechnicians and graphic workers need especial attention on occupational VOC exposure. The results from this study reinforce the need for reliable methods able to the biological monitoring as an important tool for assessing occupational exposure.

Diurnal Variation of Urinary Fabry Disease Biomarkers during Enzyme Replacement Therapy Cycles.

Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the α-galactosidase A enzyme. This enzyme cleaves the last sugar unit of glycosphingolipids, including globotriaosylceramide (Gb3), globotriaosylsphingosine (lyso-Gb3), and galabiosylceramide (Ga2). Enzyme impairment leads to substrate accumulation in different organs, vascular endothelia, and biological fluids. Enzyme replacement therapy (ERT) is a commonly used treatment. Urinary analysis of Gb3 isoforms (different fatty acid moieties), as well as lyso-Gb3 and its analogues, is a reliable way to monitor treatment. These analogues correspond to lyso-Gb3 with chemical modifications on the sphingosine moiety (−C2H4, −C2H4+O, −H2, −H2+O, +O, +H2O2, and +H2O3). The effects of sample collection time on urinary biomarker levels between ERT cycles were not previously documented. The main objective of this project was to analyze the aforementioned biomarkers in urine samples from seven Fabry disease patients (three treated males, three treated females, and one ERT-naïve male) collected twice a day (morning and evening) for 42 days (three ERT cycles). Except for one participant, our results show that the biomarker levels were generally more elevated in the evening. However, there was less variability in samples collected in the morning. No cyclic variations in biomarker levels were observed between ERT infusions.

An Improved Prediction Model for Ovarian Cancer Using Urinary Biomarkers and a Novel Validation Strategy.

This study was designed to analyze urinary proteins associated with ovarian cancer (OC) and investigate the potential urinary biomarker panel to predict malignancy in women with pelvic masses. We analyzed 23 biomarkers in urine samples obtained from 295 patients with pelvic masses scheduled for surgery. The concentration of urinary biomarkers was quantitatively assessed by the xMAP bead-based multiplexed immunoassay. To identify the performance of each biomarker in predicting cancer over benign tumors, we used a repeated leave-group-out cross-validation strategy. The prediction models using multimarkers were evaluated to develop a urinary ovarian cancer panel. After the exclusion of 12 borderline tumors, the urinary concentration of 17 biomarkers exhibited significant differences between 158 OCs and 125 benign tumors. Human epididymis protein 4 (HE4), vascular cell adhesion molecule (VCAM), and transthyretin (TTR) were the top three biomarkers representing a higher concentration in OC. HE4 demonstrated the highest performance in all samples with OC (mean area under the receiver operating characteristic curve (AUC) 0.822, 95% CI: 0.772–0.869), whereas TTR showed the highest efficacy in early-stage OC (AUC 0.789, 95% CI: 0.714–0.856). Overall, HE4 was the most informative biomarker, followed by creatinine, carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), and TTR using the least absolute shrinkage and selection operator (LASSO) regression models. A multimarker panel consisting of HE4, creatinine, CEA, and TTR presented the best performance with 93.7% sensitivity (SN) at 70.6% specificity (SP) to predict OC over the benign tumor. This panel performed well regardless of disease status and demonstrated an improved performance by including menopausal status. In conclusion, the urinary biomarker panel with HE4, creatinine, CEA, and TTR provided promising efficacy in predicting OC over benign tumors in women with pelvic masses. It was also a non-invasive and easily available diagnostic tool.

Evidence for interactions between inflammatory markers and renin-angiotensin system molecules in the occurrence of albuminuria in children with sickle cell anemia

Sickle cell anemia (SCA) is an important cause of chronic kidney disease, but its pathophysiology is not completely understood. The aim of this study was to compare inflammatory biomarkers in urine samples of SCA children with and without albuminuria, and to explore correlations with renin-angiotensin system (RAS) molecules. A cross-sectional study of 213 children selected from the Minas Gerais state cohort were assigned to two groups: Group 1–89 children with SCA who had albuminuria; Group 2–124 children with SCA and normal albuminuria matched by age and sex with group 1. A subset of 89 children was prospectively followed for a median time of 1.1 year. Inflammatory biomarkers (chemokines and cytokines) in urine were measured using cytometric beads array, and RAS molecules were measured by ELISA. Children with albuminuria had significantly higher urinary levels of IP-10/CXCL10, MCP-1/CCL2, MIG/CXCL9, IL-8/CXCL8, IL-12p70, TNF, IL-10, and IL-6 than patients with normal albuminuria. In the correlation analysis, albumin/creatinine ratio was significantly and positively correlated with IP-10/CXCL10, MCP-1/CCL2, MIG/CXCL9, IL-8/CXCL8, TNF, IL-10, and IL-6. Significant correlations were found between inflammatory and RAS molecules. In the prospective analysis, cumulative risk of persistent albuminuria was higher for children with urinary levels of IP-10/CXCL10 or IL-6 above the 50th percentile. Our data showed that inflammatory markers and RAS molecules might contribute to the occurrence of albuminuria in children with SCA, suggesting that both pathways interact in sickle cell nephropathy.

Application of Urinary Polyphenol Biomarkers Measured by Liquid Chromatography Tandem Mass Spectrometry to Assess Polyphenol Intake and Their Association with Overweight and Obesity in Free-Living Healthy Subjects

Although some polyphenol biomarkers in serum or urine have been identified by untargeted metabolomics and proved to reflect dietary polyphenol intake, only a few of them have been validated in different studies and populations with simple and reliable targeted methods. In the present study, a targeted metabolomics method by LC/MS/MS for the measurement of twenty-two polyphenol biomarkers in urine samples was established and validated to effectively assess the habitual polyphenol intake in free-living healthy Chinese subjects. Multivariate logistic regression models were used to assess relationships of biomarkers with overweight and obesity after adjusting for potential confounders. The levels of urinary polyphenol biomarkers, especially gut microbial metabolites of polyphenols, were inversely associated with overweight and obesity, and this association was more pronounced in the inflammatory groups, suggesting that it is of great importance to maintain polyphenol biomarkers at high levels or intake-sufficient polyphenols in obesity with chronic inflammation than others. The measurement of these biomarkers may offer a valid alternative or complementary addition to self-reported survey for the evaluation of polyphenol intake and investigation into their relationships with chronic disease-related endpoints in large-scale clinical and epidemiologic studies.