High-Resolution Magic-Angle-Spinning NMR in Revealing Hepatoblastoma Hallmarks

Cancer is one of the leading causes of death in children and adolescents worldwide; among the types of liver cancer, hepatoblastoma (HBL) is the most common in childhood. Although it affects only two to three individuals in a million, it is mostly asymptomatic at diagnosis, so by the time it is detected it has already advanced. There are specific recommendations regarding HBL treatment, and ongoing studies to stratify the risks of HBL, understand the pathology, and predict prognostics and survival rates. Although magnetic resonance imaging spectroscopy is frequently used in diagnostics of HBL, high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy of HBL tissues is scarce. Using this technique, we studied the alterations among tissue metabolites of ex vivo samples from (a) HBL and non-cancer liver tissues (NCL), (b) HBL and adjacent non-tumor samples, and (c) two regions of the same HBL samples, one more centralized and the other at the edge of the tumor. It was possible to identify metabolites in HBL, then metabolites from the HBL center and the border samples, and link them to altered metabolisms in tumor tissues, highlighting their potential as biochemical markers. Metabolites closely related to liver metabolisms such as some phospholipids, triacylglycerides, fatty acids, glucose, and amino acids showed differences between the tissues

Evaluation of the universality of NMR metabolic fingerprints of schizophrenia

Schizophrenia (SCZ) is a very disabling mental disorder whose molecular basis is a combination of many factors still not completely understood, with a diagnosis based on observed behavior, the person's reported experiences and reports of others that are familiar with the person, with no objective test. Also, up to date, there are no reliable markers for monitoring the SCZ. NMR-metabolomics [1] reported in 2017 bring some of the possible markers from blood serum of SCZ individuals linked strongly with known dopamine, glutamate and GABA dysfunction in SCZ. As to verify if these findings are universal, we have compared the SCZ patients from geographically different environments and cited interesting SCZ characteristics. The first set of samples was collected in Belgrade, Serbia. 14 mental health patients (50% male) with 52.86 ± 7.27 years of age had a confirmed diagnosis of SCZ. The control group of 13 healthy individuals (69% male) had none of psychotic disorders, and individuals were 23.07 ± 2.79 years of age. Blood serum samples were collected and prepared for the analysis following the published methodology [1, 2]. NMR spectra were measured on a Bruker AVANCE III spectrometer (500.26 MHz for 1H). The spectra were acquired at 298 K with 128 scans and 32 k. The serum samples were prepared and measured as triplicates. On the other side, the group of individuals from Brazil that was matched in number, age, gender and history of mental illness with individuals from Serbia was previously described [1]. 1H NMR spectra were phase and baseline corrected using MestreNova and the lactate doublet was used as the chemical shift reference. The data were binned (0.005 ppm) in a spectral range 0.50 - 9.00 ppm, while the residual HDO peak (4.50-5.00 ppm) was excluded. Then, the data were normalized by the sum equal to 1000, the variables were mean centered and PCA and PLS-DA were performed using MATLAB. It was shown that the mental health patients have clearly different blood serum metabolites when compared to the healthy ones independently from where the samples were obtained with almost identical marker set. Also, it was shown that the samples are different metabolically when Brazilian and Serbian samples were compared. 1] L. Tasic et al., Schizophrenia Research 2017, 185, 182. [2] J. Pontes et al., Analytical Methods 2017, 9, 1078

¹H NMR metabonomic analysis of crack users serum

Orientador: Ljubica TasicTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuímicaResumo: Estima-se que mais de 271 milhões de pessoas - cerca de 5,5% da população adulta mundial - consumiram drogas em 2017, das quais cerca de 18,1 milhões usaram cocaína. O crack representa uma extensa fração do uso de cocaína, já que é barata e amplamente disponível nas Américas e cada vez mais na Europa. É consumido por inalação do vapor expelido pela queima das pedras, produzindo um estalo característico que deu origem ao termo crack. É difícil estimar o número de usuários de crack, por serem marginalizados socioeconomicamente e, portanto, geralmente não são alcançados por avaliações epidemiológicas. Vários problemas de saúde física e mental são comumente relatados em usuários de crack. Apesar do conhecimento sobre a neurobiologia da dependência de crack e o número de estudos em busca de tratamentos farmacológicos eficazes estarem crescendo, não existem farmacoterapias eficazes comprovadas. Diferentes metodologias têm sido usadas para melhorar o diagnóstico da dependência de drogas ilícitas. O objetivo desse trabalho é um estudo metabonômico de usuários de crack, a partir da aplicação de técnicas quimiométricas a dados espectrais de RMN de ¹H de amostras de soro, buscando a identificação de biomarcadores relacionados ao vício em crack (comparando usuários e voluntários saudáveis) e ao tratamento (comparando mulheres pacientes antes e após o tratamento). Foram utilizadas amostras de soro de 44 usuários de crack (pacientes) e de 44 voluntários saudáveis (controles). Para a avaliação do tratamento, foram utilizadas amostras de soro de pacientes internadas em processo de ressocialização em um centro para mulheres com dependência de drogas e álcool. As amostras de soro foram analisadas por RMN de ¹H utilizando as sequências de pulsos de supressão de água, Watergate, e de filtro de T2, CPMG. Para classificação dos indivíduos nos grupos pacientes e controle o modelo PLS-DA obteve 89,8% e 93,2 % de exatidão na validação cruzada total usando 4 e 7 variáveis latentes, respectivamente. A área sob a curva ROC do modelo LDA foi de 0,98. As diferenças observadas na tirosina, histidina, lactato e acil carnitinas, apontam para efeitos do crack no cérebro, já que podem estar ligados a funções cerebrais alteradas. Particularmente o lactato e as acil carnitinas, podem ser associados à condição nutricional dos pacientes. As análises estatísticas dos espectros de soro das pacientes antes e depois do tratamento de três semanas indicam que glicose e glicina estão presentes em maior quantidade nas amostras de soro das pacientes antes do tratamento, enquanto a quantidade de lipídios variou[TBdC1] [TBdC2] , o que pode ser consequência da mudança de hábitos alimentares ou dos níveis de estresse apresentados pelas usuárias durante o período de abstinência e tratamento. É provável que essa falta de padrão nos espectros dos metabólitos seja um reflexo da heterogeneidade dos grupos, uma vez que a responsividade das pacientes ao tratamento com os medicamentos atualmente utilizados depende de inúmeros fatores, desde negligências e abusos sofridos na infância até níveis de estresse durante o período de internação, além da falta de medicamento específico e eficaz para o tratamento de vício em crackAbstract: It is estimated that more than 271 million people - about 5.5% of the world's adult population - used drugs in 2017, of which about 18.1 million used cocaine. Crack represents an extensive fraction of cocaine use, as it is cheap and widely available in the Americas and increasingly in Europe. It is consumed by inhaling the vapor expelled by burning the stones, producing a characteristic crack that gave rise to the term crack. It is difficult to estimate the actual number of crack users, as they are marginalized socioeconomically and, therefore, are generally not included in the epidemiological assessments. Several physical and mental health problems are commonly reported in crack users. Despite the growing knowledge about the neurobiology of crack addiction and the growing number of studies looking for effective pharmacological treatments, there are no proven effective pharmacotherapies for crack addiction to date. Different methodologies have been used to improve the diagnosis of illicit drug addiction. The objective of this work is a metabonomic study of crack users, from the application of chemometric techniques to ¹H NMR spectral data from serum samples, seeking to identify biomarkers related to crack addiction (users versus healthy volunteers) and to treatment (comparing crack addicted women before and after treatment). For classification of individuals in the patient and control groups, the PLS-DA model obtained 89.8% and 93.2% accuracy in the total cross-validation using 4 and 7 latent variables, respectively. The area under the ROC curve of the LDA model was 0.98. The differences observed in tyrosine, histidine, lactate and acyl carnitines, point to the effects of crack in the brain, since they may be linked to altered brain functions. Particularly lactate and acyl carnitines, can be associated with the nutritional condition of patients. Statistical analyzes of the patients' spectra before and after the three-week treatment indicate that glucose and glycine are present in greater quantities in the patient's serum samples before treatment, while the amount of lipids varied, which may be a consequence of the change in eating habits or stress levels presented by users during the abstinence period. It is likely that this lack of pattern in the spectrum of metabolites is a reflection of the heterogeneity of the groups, since the responsiveness of patients to treatment with the drugs currently used depends on numerous factors, from neglect and abuse suffered in childhood to levels of stress during the hospitalization period, in addition to the lack of specific and effective medication for the treatment of crack addictionDoutoradoQuímica OrgânicaDoutora em Ciências001CAPE

Effects of psychostimulants and antipsychotics on serum lipids in an animal model for schizophrenia

Schizophrenia (SCZ) treatment is essentially limited to the use of typical or atypical antipsychotic drugs, which suppress the main symptoms of this mental disorder. Metabolic syndrome is often reported in patients with SCZ under long-term drug treatment, but little is known about the alteration of lipid metabolism induced by antipsychotic use. In this study, we evaluated the blood serum lipids of a validated animal model for SCZ (Spontaneously Hypertensive Rat, SHR), and a normal control rat strain (Normotensive Wistar Rat, NWR), after long-term treatment (30 days) with typical haloperidol (HAL) or atypical clozapine (CLZ) antipsychotics. Moreover, psychostimulants, amphetamine (AMPH) or lisdexamfetamine (LSDX), were administered to NWR animals aiming to mimic the human first episode of psychosis, and the effects on serum lipids were also evaluated. Discrepancies in lipids between SHR and NWR animals, which included increased total lipids and decreased phospholipids in SHR compared with NWR, were similar to the differences previously reported for SCZ patients relative to healthy controls. Administration of psychostimulants in NWR decreased omega-3, which was also decreased in the first episode of psychosis of SCZ. Moreover, choline glycerophospholipids allowed us to distinguish the effects of CLZ in SHR. Thus, changes in the lipid metabolism in SHR seem to be reversed by the long-term treatment with the atypical antipsychotic CLZ, which was under the same condition described to reverse the SCZ-like endophenotypes of this validated animal model for SCZ. These data open new insights for understanding the potential influence of the treatment with typical or atypical antipsychotics on circulating lipids. This may represent an outcome effect from metabolic pathways that regulate lipids synthesis and breakdown, which may be reflecting a cell lipids dysfunction in SCZ.Instituto de Química, Universidade Estadual de Campinas (UNICAMP), Campinas 13083-970, BrazilNational Institute for Translational Medicine (INCT-TM, CNPq), Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP), São Paulo 14049-900, BrazilDepartamento de Farmacologia, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo 04044-020, Brazil.Departamento de Farmacologia, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), São Paulo 04044-020, Brazil

Lipidomics by Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography–High-Resolution Mass Spectrometry in Osteosarcoma: A Pilot Study

Cancer is a complex disease that can also affect the younger population; however, it is responsible for a relatively high mortality rate of children and youth, especially in low- and middle-income countries (LMICs). Besides that, lipidomic studies in this age range are scarce. Therefore, we analyzed blood serum samples from young patients (12 to 35 years) with bone sarcoma (osteosarcoma) and compared their lipidomics to the ones from the control group of samples, named healthy control (HC group), using NMR and LC-MS techniques. Furthermore, differences in the lipidomic profiles between OS patients with and without metastasis indicate higher glycerophosphocholine (GPC) and glycerophospholipid (GPL) levels in osteosarcoma and increased cholesterol, choline, polyunsaturated fatty acids (PUFAs), and glycerols during the metastasis. These differences, detected in the peripheral blood, could be used as biomarkers for liquid biopsy