Metabolomics contributions to targeted and untargeted clinical analysis by chromatography and mass spectrometry

La investigación desarrollada en esta Tesis Doctoral se centró en realizar contribuciones en el ámbito del análisis clínico a través de estrategias metabolómicas tanto orientadas como globales en diferentes tipos de muestras biológicas mediante cromatografía de líquidos (LC) y espectrometría de masas (MS). Para ello primeramente se revisó exhaustiva y críticamente la bibliografía para conocer el estado del tratamiento de la orina como muestra y de los métodos de análisis de aire exhalado condensado, dos de los biofluidos utilizados en el desarrollo de esta Tesis. Además, se optimizaron métodos de análisis orientado para mejorar la cuantificación tanto de compuestos con potencial biomarcador como de fármacos y sus metabolitos para su aplicación en el diagnóstico y seguimiento de enfermedades o tratamientos. También se analizaron de forma global biofluidos para, (a) estudiar y optimizar el tratamiento de una muestra escasamente utilizada en el área clínica (el aire exhalado condensado –EBC), (b) identificar metabolitos con potencial predictivo para ayudar en el diagnóstico del cáncer de próstata utilizando la orina como muestra, y (c) mejorar y acelerar el tratamiento de datos a través de herramientas quimiométricas desarrolladas para combinar en una única matriz los datos obtenidos mediante ionización positiva y negativa en espectrometría de masas

Development of Bioanalytical Methods for Clinical Applications and Drug Screening

In the past decade, bioanalytical method development has become an integral part of clinical diagnosis, biomarker discovery, and drug discovery and development. The new and emerged bioanalytical technologies allow the quantitative and qualitative analysis of small molecules and biomolecules with high sensitivity and specificity. Specifically, the bioanalytical methods based on LC-MS and methylation-specific PCR are well suited for detecting low-abundance metabolites, proteins, and DNA in biofluids and tissues for biomarker investigation. They offer great clinical promises for early diseases diagnosis and therapeutic interventions. Besides, the LC-MS/MS quantitative method is essential for the estimation of pharmacokinetic and toxicological properties in drug screening.In this work, modern bioanalytical technologies, together with their applications from biomarker discovery and validation in metabonomics, genomics and proteomics to drug discovery, were reviewed. Dependent on the type of molecules analyzed, different methods were established to achieve accurate and reliable detection. LC-MS/(MS) methods were developed and validated for quantitative analysis of bile acids and anti-cancer agent JCC76. The former has been successfully applied in a clinical study for the diagnosis of inflammatory bowel diseases and the latter has been utilized in a pharmacokinetics study for drug screening and optimization. In terms of proteomics profiling, a LC-MS/MS method was demonstrated for comparative analysis of serum peptides with the successful identification of a potential biomarker for ovarian cancer. Lastly, a comprehensive DNA methylation profiling for hepatocellular carcinoma was conducted through methylation-specific PCR methods. These methods enabled sensitive and specific detection of DNA hypermethylation on several tumor-associated genes. In addition, this work discussed a major challenge of matrix effect in quantitative method development. Possible solutions were proposed for matrix effect prevention and troublesh

Development of Bioanalytical Methods for Clinical Applications and Drug Screening

In the past decade, bioanalytical method development has become an integral part of clinical diagnosis, biomarker discovery, and drug discovery and development. The new and emerged bioanalytical technologies allow the quantitative and qualitative analysis of small molecules and biomolecules with high sensitivity and specificity. Specifically, the bioanalytical methods based on LC-MS and methylation-specific PCR are well suited for detecting low-abundance metabolites, proteins, and DNA in biofluids and tissues for biomarker investigation. They offer great clinical promises for early diseases diagnosis and therapeutic interventions. Besides, the LC-MS/MS quantitative method is essential for the estimation of pharmacokinetic and toxicological properties in drug screening.In this work, modern bioanalytical technologies, together with their applications from biomarker discovery and validation in metabonomics, genomics and proteomics to drug discovery, were reviewed. Dependent on the type of molecules analyzed, different methods were established to achieve accurate and reliable detection. LC-MS/(MS) methods were developed and validated for quantitative analysis of bile acids and anti-cancer agent JCC76. The former has been successfully applied in a clinical study for the diagnosis of inflammatory bowel diseases and the latter has been utilized in a pharmacokinetics study for drug screening and optimization. In terms of proteomics profiling, a LC-MS/MS method was demonstrated for comparative analysis of serum peptides with the successful identification of a potential biomarker for ovarian cancer. Lastly, a comprehensive DNA methylation profiling for hepatocellular carcinoma was conducted through methylation-specific PCR methods. These methods enabled sensitive and specific detection of DNA hypermethylation on several tumor-associated genes. In addition, this work discussed a major challenge of matrix effect in quantitative method development. Possible solutions were proposed for matrix effect prevention and troublesh

Development of Bioanalytical Methods for Clinical Applications and Drug Screening

In the past decade, bioanalytical method development has become an integral part of clinical diagnosis, biomarker discovery, and drug discovery and development. The new and emerged bioanalytical technologies allow the quantitative and qualitative analysis of small molecules and biomolecules with high sensitivity and specificity. Specifically, the bioanalytical methods based on LC-MS and methylation-specific PCR are well suited for detecting low-abundance metabolites, proteins, and DNA in biofluids and tissues for biomarker investigation. They offer great clinical promises for early diseases diagnosis and therapeutic interventions. Besides, the LC-MS/MS quantitative method is essential for the estimation of pharmacokinetic and toxicological properties in drug screening.In this work, modern bioanalytical technologies, together with their applications from biomarker discovery and validation in metabonomics, genomics and proteomics to drug discovery, were reviewed. Dependent on the type of molecules analyzed, different methods were established to achieve accurate and reliable detection. LC-MS/(MS) methods were developed and validated for quantitative analysis of bile acids and anti-cancer agent JCC76. The former has been successfully applied in a clinical study for the diagnosis of inflammatory bowel diseases and the latter has been utilized in a pharmacokinetics study for drug screening and optimization. In terms of proteomics profiling, a LC-MS/MS method was demonstrated for comparative analysis of serum peptides with the successful identification of a potential biomarker for ovarian cancer. Lastly, a comprehensive DNA methylation profiling for hepatocellular carcinoma was conducted through methylation-specific PCR methods. These methods enabled sensitive and specific detection of DNA hypermethylation on several tumor-associated genes. In addition, this work discussed a major challenge of matrix effect in quantitative method development. Possible solutions were proposed for matrix effect prevention and troublesh

Urinary metabolic signatures detect recurrences in non-muscle invasive bladder cancer

Patients with non-muscle invasive bladder cancer (NMIBC) undergo lifelong monitoring based on repeated cystoscopy and urinary cytology due to the high recurrence rate of this tumor. Nevertheless, these techniques have some drawbacks, namely, low accuracy in detection of low-grade tumors, omission of pre-neoplastic lesions and carcinomas in situ (CIS), invasiveness, and high costs. This work aims to identify a urinary metabolomic signature of recurrence by proton Nuclear Magnetic Resonance (1H NMR) spectroscopy for the follow-up of NMIBC patients. To do this, changes in the urinary metabolome before and after transurethral resection (TUR) of tumors are analyzed and a Partial Least Square Discriminant Analysis (PLS-DA) model is developed. The usefulness of this discriminant model for the detection of tumor recurrences is assessed using a cohort of patients undergoing monitoring. The trajectories of the metabolomic profile in the follow-up period provide a negative predictive value of 92.7% in the sample classification. Pathway analyses show taurine, alanine, aspartate, glutamate, and phenylalanine perturbed metabolism associated with NMIBC. These results highlight the potential of 1H NMR metabolomics to detect bladder cancer (BC) recurrences through a non-invasive approach

Identification of Apo-A1 as a biomarker for early diagnosis of bladder transitional cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Bladder transitional cell carcinoma (BTCC) is the fourth most frequent neoplasia in men, clinically characterized by high recurrent rates and poor prognosis. Availability of urinary tumor biomarkers represents a convenient alternative for early detection and disease surveillance because of its direct contact with the tumor and sample accessibility.</p> <p>Results</p> <p>We tested urine samples from healthy volunteers and patients with low malignant or aggressive BTCC to identify potential biomarkers for early detection of BTCC by two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) and bioinformatics analysis. We observed increased expression of five proteins, including fibrinogen (Fb), lactate dehydrogenase B (LDHB), apolipoprotein-A1 (Apo-A1), clusterin (CLU) and haptoglobin (Hp), which were increased in urine samples of patients with low malignant or aggressive bladder cancer. Further analysis of urine samples of aggressive BTCC showed significant increase in Apo-A1 expression compared to low malignant BTCC. Apo-A1 level was measured quantitatively using enzyme-linked immunosorbent assay (ELISA) and was suggested to provide diagnostic utility to distinguish patients with bladder cancer from controls at 18.22 ng/ml, and distinguish patients with low malignant BTCC from patients with aggressive BTCC in two-tie grading system at 29.86 ng/ml respectively. Further validation assay showed that Apo-A1 could be used as a biomarker to diagnosis BTCC with a sensitivity and specificity of 91.6% and 85.7% respectively, and classify BTCC in two-tie grading system with a sensitivity and specificity of 83.7% and 89.7% respectively.</p> <p>Conclusion</p> <p>Taken together, our findings suggest Apo-A1 could be a potential biomarker related with early diagnosis and classification in two-tie grading system for bladder cancer.</p

Assessment of a Capsule for Easy Urine Proteome Collection at Home

Urine as a biological sample contains a wealth of information with great significance for med-ical diagnosis while also benefiting from an easy and non-invasive collection procedure. Thus, urine has become one of the standard samples for diagnosing multiple conditions and for proteomics-based biomarker discovery studies. In recent years, our group has developed an approach for Bladder cancer (BCa) diagnosis and patient monitoring based on proteomics, using the dysregulation of the urinary proteome to gain information regarding the patient’s state, such as tumour diagnosis and stage. Furthermore, analysing multiple urine samples from different time points provides valuable insight into the patient's treatment response and re-covery. However, monitoring is impaired by the lack of sample collection at the hospital. Thus, our goal was to develop a new easy-to-use method of urinary proteome collection so that patients could collect their samples at home. Thus, the urine proteome was collected using syringe filters and handled with a standard shotgun proteomics protocol. Multiple filter mem-branes were tested, from which the molecular weight cut-off membranes were the most effi-cient for our proposal. A proof of concept was conducted with BCa patient samples. The Filter Aided Sample Preparation (FASP) protocol was used as a control for the results obtained with our new approach. Our results show that the urine proteome can be collected and treated using our filter approach, although further research is needed to increase digestion efficiency to standard protocol levels. We propose syringe filtering as an easy approach to follow-up BCa patients and potentially any other diseases that can be assessed using the urine proteome.A urina enquanto amostra biológica contém informação extremamente relevante para o diagnóstico de diversas condições médicas, sendo recolhida de um modo fácil e não invasivo, tornando-se uma das amostras biológicas ideais para diagnóstico de condições médicas e para estudos de proteómica baseada em espectrometria de massa. Recentemente, o nosso grupo desenvolveu uma metodologia para diagnóstico e monitorização de pacientes de cancro da bexiga (BCa), onde a desregulação do proteoma urinário permite o diagnóstico e identificação de estadio do tumor. Adicionalmente, ao analisar amostras recolhidas ao longo do tempo, é possível fazer o seguimento do paciente, obtendo informação acerca da sua resposta ao tra-tamento. Esta monitorização é, contudo, dificultada devido aos grandes intervalos de tempo entre recolhas. Sendo assim, o nosso objetivo foi desenvolver uma nova metodologia para recolha do proteoma urinário, de uso autónomo pelo paciente. Assim, o proteoma urinário foi recolhido usando filtros de seringa e analisado com um protocolo de shotgun proteomics. Foram testadas várias membranas, sendo que as de separação por tamanho molecular foram as mais eficientes na recolha do proteoma. Foi ainda feito um proof of concept com amostras de pacientes de BCa onde o protocolo de Filter Aided Sample Preparation (FASP), foi usando como controlo para os resultados obtidos com a nova metodologia. Os resultados obtidos demonstram que o proteoma urinário pode ser eficazmente recolhido usando estes filtros, contudo a técnica requer otimizações para alcançar níveis de digestão proteica similares aos dos protocolos standard. Propomos filtros de seringa como uma técnica fácil para monitorizar pacientes de BCa, e potencialmente outras doenças a partir do proteoma urinário

Determination of Serum CA125 and evaluate its efficiency as screening tool For Early Detection of Ovarian Tumors

Epithelial ovarian cancer is the leading cause of cancer deaths in women. To date, an effective screening tool for ovarian cancer has not been identified Several clinical and biological factors including serum cancer antigen 125 (CA- 125) have been assessed for prognostic and predictive relevance CA-125 is an epithelial marker derived from coelomic epithelium. It is elevated in 90% of advanced ovarian cancers and in 50% of early ovarian cancers while 20% of ovarian cancers have low or no expression of CA- 125 CA-125 concentrations were measured by Mini Vidas test (VIDAS CA125 II / BIOMERIEUX / France). The median CA-125 levels were significantly higher in the sera of ovarian cancer patients than in those with benign tumors and in healthy controls. However in correlation with stages the results showed that Patients with stage II have highly significant differences in level of serum CA125 compare with stage I in and stage III.CA125 showed low sensitivity to detect stage I carcinoma of the ovary which limits its value as an initial screening tool therefore combining of CA125 with other markers might enable improved early detection of ovarian cancer as compared with use of this marker alone

Shed urinary ALCAM is an independent prognostic biomarker of three-year overall survival after cystectomy in patients with bladder cancer.

Proteins involved in tumor cell migration can potentially serve as markers of invasive disease. Activated Leukocyte Cell Adhesion Molecule (ALCAM) promotes adhesion, while shedding of its extracellular domain is associated with migration. We hypothesized that shed ALCAM in biofluids could be predictive of progressive disease. ALCAM expression in tumor (n = 198) and shedding in biofluids (n = 120) were measured in two separate VUMC bladder cancer cystectomy cohorts by immunofluorescence and enzyme-linked immunosorbent assay, respectively. The primary outcome measure was accuracy of predicting 3-year overall survival (OS) with shed ALCAM compared to standard clinical indicators alone, assessed by multivariable Cox regression and concordance-indices. Validation was performed by internal bootstrap, a cohort from a second institution (n = 64), and treatment of missing data with multiple-imputation. While ALCAM mRNA expression was unchanged, histological detection of ALCAM decreased with increasing stage (P = 0.004). Importantly, urine ALCAM was elevated 17.0-fold (P &lt; 0.0001) above non-cancer controls, correlated positively with tumor stage (P = 0.018), was an independent predictor of OS after adjusting for age, tumor stage, lymph-node status, and hematuria (HR, 1.46; 95% CI, 1.03-2.06; P = 0.002), and improved prediction of OS by 3.3% (concordance-index, 78.5% vs. 75.2%). Urine ALCAM remained an independent predictor of OS after accounting for treatment with Bacillus Calmette-Guerin, carcinoma in situ, lymph-node dissection, lymphovascular invasion, urine creatinine, and adjuvant chemotherapy (HR, 1.10; 95% CI, 1.02-1.19; P = 0.011). In conclusion, shed ALCAM may be a novel prognostic biomarker in bladder cancer, although prospective validation studies are warranted. These findings demonstrate that markers reporting on cell motility can act as prognostic indicators