Investigation of urinary volatile organic metabolites as potential cancer biomarkers by solid-phase microextraction in combination with gas chromatography-mass spectrometry

BACKGROUND: Non-invasive diagnostic strategies aimed at identifying biomarkers of cancer are of great interest for early cancer detection. Urine is potentially a rich source of volatile organic metabolites (VOMs) that can be used as potential cancer biomarkers. Our aim was to develop a generally reliable, rapid, sensitive, and robust analytical method for screening large numbers of urine samples, resulting in a broad spectrum of native VOMs, as a tool to evaluate the potential of these metabolites in the early diagnosis of cancer. METHODS: To investigate urinary volatile metabolites as potential cancer biomarkers, urine samples from 33 cancer patients (oncological group: 14 leukaemia, 12 colorectal and 7 lymphoma) and 21 healthy (control group, cancer-free) individuals were qualitatively and quantitatively analysed. Dynamic solid-phase microextraction in headspace mode (dHS-SPME) using a carboxenpolydimethylsiloxane (CAR/PDMS) sorbent in combination with GC-qMS-based metabolomics was applied to isolate and identify the volatile metabolites. This method provides a potential non-invasive method for early cancer diagnosis as a first approach. To fulfil this objective, three important dHS-SPME experimental parameters that influence extraction efficiency (fibre coating, extraction time and temperature of sampling) were optimised using a univariate optimisation design. The highest extraction efficiency was obtained when sampling was performed at 501C for 60min using samples with high ionic strengths (17% sodium chloride, wv 1) and under agitation. RESULTS: A total of 82 volatile metabolites belonging to distinct chemical classes were identified in the control and oncological groups. Benzene derivatives, terpenoids and phenols were the most common classes for the oncological group, whereas ketones and sulphur compounds were the main classes that were isolated from the urine headspace of healthy subjects. The results demonstrate that compound concentrations were dramatically different between cancer patients and healthy volunteers. The positive rates of 16 patients among the 82 identified were found to be statistically different (Po0.05). A significant increase in the peak area of 2-methyl3-phenyl-2-propenal, p-cymene, anisole, 4-methyl-phenol and 1,2-dihydro-1,1,6-trimethyl-naphthalene in cancer patients was observed. On average, statistically significant lower abundances of dimethyl disulphide were found in cancer patients. CONCLUSIONS: Gas chromatographic peak areas were submitted to multivariate analysis (principal component analysis and supervised linear discriminant analysis) to visualise clusters within cases and to detect the volatile metabolites that are able to differentiate cancer patients from healthy individuals. Very good discrimination within cancer groups and between cancer and control groups was achieved.

Seasonal changes in fecal testosterone concentrations and their relationship to the reproductive behavior, antler cycle and grouping patterns in free-ranging male Pampas deer (Ozotoceros bezoarticus bezoarticus)

The purpose of this study was to validate noninvasive endocrine monitoring techniques for Pampas deer and to evaluate seasonal changes in testicular steroidogenic activity and their correlation to reproductive behavior, antler cycle and group size. Thus, fecal samples, behavioral data and observations of antler status were collected at monthly intervals during 1 year from free-ranging Pampas deer stags (three radio-collared individuals and 15 random individuals) living in Emas National Park, Brazil (18 degrees S latitude). Fecal steroids were extracted using 80% methanol and steroid concentrations were quantified by a commercial enzyme immunoassay (EIA). Fecal testosterone concentrations peaked in December-January (summer), March (early autumn) and in August-September (winter-spring), with minimal values from April-July. Reproductive behavior had two peaks, the first in December-January, characterized by predominately anogenital sniffing, flehmen, urine sniffing, chasing and mounting behavior, and the second peak in July-September (behavior primarily related to gland marking). There were significant correlations between fecal testosterone and reproductive behavior (r = 0.490), and between fecal testosterone and antler phases (r = 0.239). Antler casting and regrowth occurred under low testosterone concentrations, whereas velvet shedding was associated with high concentrations of testosterone. We inferred that Pampas deer stags exhibited a seasonal cycle that modulated sexual behavior and the antler cycle, and we concluded that fecal steroid analysis was a practical and reliable non-invasive method for the evaluation of the endocrine status of free-ranging Pampas deer. (c) 2004 Elsevier B.V. All rights reserved.

Associação entre espessura da pele e densidade óssea em mulheres adultas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Úlceras crônicas dos membros inferiores: avaliação pela fotografia digital

OBJETIVO: Avaliar o desempenho da análise de imagem digital na estimativa da área acometida pelas úlceras crônicas dos membros inferiores. MÉTODOS: Estudo prospectivo em que foram mensuradas úlceras empregando o método planimétrico clássico, utilizando desenho dos seus contornos em filme plástico transparente, medida sua área posteriormente por folha milimetrada. Esses valores foram utilizados como padrão para a comparação com a estimativa de área pelas fotografias digitais padronizadas das úlceras e dos desenhos das mesmas em filme plástico. Para criar um referencial de conversão dos pixels em milímetros, foi empregado um adesivo com tamanho conhecido, adjacente à úlcera. RESULTADOS: foram avaliadas 42 lesões em 20 pacientes portadores de úlceras crônicas de membros inferiores. As áreas das úlceras variaram de 0,24 a 101,65cm². Observou-se forte correlação entre as medidas planimétricas e as fotos das úlceras (R²=0,86 p<0,01), porém a correlação das medidas planimétricas com as fotos digitais dos desenhos das úlceras foi ainda maior (R²=0,99 p<0,01). CONCLUSÃO: A fotografia digital padronizada revelou-se método rápido, preciso e não-invasivo capaz de estimar a área afetada por úlceras. A avaliação das medidas fotográficas dos contornos das úlceras deve ser preferida em relação à análise de sua fotografia direta.

Saliva: reflection of the body

Saliva has become an important resource for evaluating physiological and pathological conditions in humans. The use of saliva has many advantages, including the simple and non-invasive method of collection and its easy, low-cost storage. With the addition of modern techniques and chemical instrumentation equipment, there has been an increase in its use for laboratory investigations, applicable for basic and clinical analyses in the fields of medicine and dentistry. The value of these methods for the diagnosis of oral and systemic diseases has been the subject of study by several researchers with the aim of increasing its use alongside complementary exams. (C) 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Blood lactate response and critical speed in swimmers aged 10-12 years of different standards

It has previously been shown that measurement of the critical speed is a non-invasive method of estimating the blood lactate response during exercise. However, its validity in children has yet to be demonstrated. The aims of this study were: (1) to verify if the critical speed determined in accordance with the protocol of Wakayoshi et al. is a non-invasive means of estimating the swimming speed equivalent to a blood lactate concentration of 4 mmol . l(-1) in children aged 10-12 years; and (2) to establish whether standard of performance has an effect on its determination. Sixteen swimmers were divided into two groups: beginners and trained. They initially completed a protocol for determination of speed equivalent to a blood lactate concentration of 4 mmol . l(-1). Later, during training sessions, maximum efforts were swum over distances of 50, 100 and 200 m for the calculation of the critical speed. The speeds equivalent to a blood lactate concentration of 4 mmol . l(-1) (beginners = 0.82 +/- 0.09 m . s(-1), trained = 1.19 +/- 0.11 m . s(-1); mean +/- s) were significantly faster than the critical speeds (beginners = 0.78 +/- 0.25 m . s(-1), trained = 1.08 +/- 0.04 m . s(-1)) in both groups. There was a high correlation between speed at a blood lactate concentration of 4 mmol . l(-1) and the critical speed for the beginners (r = 0.96, P < 0.001), but not for the trained group (r = 0.60, P > 0.05). The blood lactate concentration corresponding to the critical speed was 2.7 +/- 1.1 and 3.1 +/- 0.4 mmol . l(-1) for the beginners and trained group respectively. The percent difference between speed at a blood lactate concentration of 4 mmol . l(-1) and the critical speed was not significantly different between the two groups. At all distances studied, swimming performance was significantly faster in the trained group. Our results suggest that the critical speed underestimates swimming intensity corresponding to a blood lactate concentration of 4 mmol . l(-1) in children aged 10-12 years and that standard of performance does not affect the determination of the critical speed.

Classification of normal swallowing and oropharyngeal dysphagia using wavelet

Oropharyngeal dysphagia is characterized by any alteration in swallowing dynamics which may lead to malnutrition and aspiration pneumonia. Early diagnosis is crucial for the prognosis of patients with dysphagia, and the best method for swallowing dynamics assessment is swallowing videofluoroscopy, an exam performed with X-rays. Because it exposes patients to radiation, videofluoroscopy should not be performed frequently nor should it be prolonged. This study presents a non-invasive method for the pre-diagnosis of dysphagia based on the analysis of the swallowing acoustics, where the discrete wavelet transform plays an important role to increase sensitivity and specificity in the identification of dysphagic patients. (C) 2008 Elsevier B.V. All rights reserved.

Estudo da pressão arterial pelo método indireto oscilométrico (petmap®) em cães domésticos não anestesiados

O estudo das pressões arteriais sistólica, média, diastólica e da frequência cardíaca, pelo método indireto oscilométrico (petmap®), foi realizado em 150 cães atendidos pelo Serviço de Clínica Médica de Pequenos Animais da FMVZ - Unesp/Botucatu. Investigou-se a influência de fatores como presença do proprietário, estado de saúde, diagnóstico de doença renal, raça, idade, sexo, decúbito, contenção, fluidoterapia, condição corpórea, temperamento, atividade física, dieta e atitude associados ou não à elevação da pressão arterial. Dos 150 cães, 34% encontravam-se sob a categoria de risco mínimo para o desenvolvimento de lesões hipertensivas, 14,6% com hipertensão branda, 22,6% com hipertensão moderada e 28,66%, com hipertensão grave. Houve influência, dos fatores analisados, na elevação da pressão arterial de acordo com a categoria de risco.

Comparação entre limiar anaeróbio determinado por variáveis ventilatórias e pela resposta do lactato sanguíneo em ciclistas

Many investigations have shown that the coincidence between the ventilatory thresholds and those thresholds using the lactate response does not happen all of the time, suggesting that there is no relationship between the cause-effect between these phenomena. Thus, the present study had as main purpose to compare and correlate the Oxygen consumption (V̇O 2), the power (W), and the heart rate (HR) values attained using protocols to determine the Ventilatory Threshold (VT) and the Individual Anaerobic Threshold (IAT). The sampling was constituted by eight State and National level cyclists (age: 27.88 ± 8.77 years; body mass: 65.19 ± 4.40 kg; height: 169.31 ± 5,77 cm). The IAT was determined starting from a three minutes 50 W warm up with progressive increases of 50 W.3min -1 up to achieving the voluntary exhaustion, when the blood was collected in the last 20 seconds of each phase, and during the recovering period. In order to determine the VT, it was used the same protocol used to determine the IAT, but without performing the blood collection. The VT was identified through the changes in the pulmonary ventilation, as well as of the ventilatory equivalent of the O 2 and CO 2. The t-Student test showed no significant statistical difference in any of the attained variables. The associations found were high and significant. The V̇O 2 (ml.kg -1.min. -1), P (W), and HR (bpm) corresponding to the VT and IAT, as well as the associations between variables were respectively: 48.00 ± 3.82 vs. 48.08 ± 3.71 (r = 0.90); 256.25 ± 32.04 vs. 246.88 ± 33.91 (r = 0.84); 173.75 ± 9.18 vs. 171.25 ± 12.02 (r = 0.97). According to the results attained, it can be concluded that the IAT and the VT produce similar V̇O 2, W, and HR values, favoring the adoption of the VT because it is a non-invasive method to determine the anaerobic threshold in cyclists.

Correlações entre os valores de lactato mínimo e potência anaeróbia com a potência crítica e a capacidade de trabalho anaeróbio obtidas por método invasivo e não invasivos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Determinação dos perfis de estrógenos e progestinas fecais durante o ciclo estral e gestação de paca (Cuniculus paca)

Pós-graduação em Cirurgia Veterinária - FCAV

Eletrofisiologia da visão em modelo experimental de ratos com diabetes e hipotireoidismo

O diabetes mellitus constitui um problema de saúde pública mundial devido ao seu potencial de morbidade e mortalidade. Esta patologia é caracterizada como uma desordem metabólica com hiperglicemia crônica, resultado de uma deficiência absoluta ou relativa na secreção e/ou ação da insulina. Associado a esse quadro, problemas com a tireoide são frequentemente encontrados em pacientes acometidos por diabetes, principalmente com o avançar da idade. Além disso, há um corpo de conhecimento que descreve que tanto o diabetes quanto o hipotiroidismo provocam isoladamente perdas visuais, no entanto, não há muitas publicações acerca da fisiologia envolvendo em conjunto ambas patologias com as complicações visuais. O presente trabalho objetivou investigar questões relevantes para uma compreensão mais detalhada do quadro de evolução do comprometimento retiniano em modelos animais acometidos concomitantemente por duas doenças: diabetes e hipotireoidismo. Para isso, foram utilizados 50 ratos Wistar machos (Rattus norvegicus), com dois meses de idade, pesando entre 80 a 120 gramas, divididos em quatro grupos: um grupo controle sem procedimentos; um grupo com hipotireoidismo optando por um modelo experimental de tireoidectomia bilateral; um grupo com diabetes, com aplicação de 200 mg/Kg a 2% de Aloxana; e dois grupos com as duas patologias, adotando ambos os procedimentos experimentais, alterando a sequência das patologias em questão. Nos grupos estudados foi utilizado para avaliar as alterações visuais, um método não invasivo amplamente utilizado no meio acadêmico que é o eletrorretinograma (ERG) em diferentes períodos de 30, 45 e 60 dias. Assim, encontramos uma diminuição da amplitude média da onda-a dos animais nos grupos com diabetes e com as duas patologias em todos os registros realizados, os que apresentaram maiores diferenças estatísticas foram em relação ao registro de estocópica máxima, como também diferença no potencial oscilatório de ambos os grupos com as duas patologias. Os resultados obtidos dão suporte à hipótese que as duas patologias concomitantes (diabetes e hipotireoidismo) diminuem significativamente as respostas do ERG.

Investigação de Leishmania SP em carrapatos de cães de bairros de Imperatriz-MA, através da Reação em Cadeia da Polimerase (PCR)

A Leishmaniose é uma doença causada pelo protozoário Leishmania sp, podendo acometer homem e animais dependendo da espécie do parasita, São transmitidos pelos flebotomíneos fêmeas, insetos do gênero Lutzomyia, que ao exercer o hematofagismo inoculam as formas promastigota infectantes, mas recentemente, tem sido levantado hipóteses sobre a transmissão por carrapatos. Segundo a vigilância epidemiológica de Imperatriz-MA a cidade é endêmica tanto para Leishmaniose Tegumentar (LT) quanto para a Leishmaniose Visceral (LV). Este trabalho teve como objetivo principal investigar a presença de DNA de Leishmania sp em carrapatos coletados de cães atendidos em petshop e Centro de Controle de Zoonoses do município de Imperatriz utilizando a técnica de PCR. O DNA foi extraído a partir de 640 carrapatos fêmeas e testadas utilizando o primer que amplifica o gene de mini-exon de Leishmania sp. Os carrapatos foram coletados de 41 cães de diferentes bairros da cidade de Imperatriz. A maioria dos carrapatos foram identificados como Rhipicephalus sanguineus. Os seguintes sinais clínicos sugestivos de leishmaniose foram observados nos cães: onicogrifose em 53,65% (22/41); úlceras em 63,41% (26/41), a perda de cabelo e inapetência em 39,02% (16/41). Cento e setenta carrapatos (26,56%) coletados de 16 cães apresentaram DNA de Leishmania do subgênero Viannia, responsável pela forma cutânea da doença. Não foi detectado nenhum DNA de Leishmania infantum chagasi. Carrapatos infectados foram coletados de ambos os cães sintomáticos e assintomáticos. Embora ainda não tenha sido demonstrado que os carrapatos possam transmitir Leishmania aos cães sob condições naturais, o resultado deste estudo tem vários aspectos importantes, pois é um método não-invasivo de detecção, capaz de diferenciar os grupos de parasitas em circulação, em especial se os animais não têm lesões, pode ser um indicador biológico em locais onde não é feito uma investigação sorológica e nem entomológica, podendo dar suporte aos programas de vigilância de saúde local.

Characterisation of the methylation pattern in the intragenic CpG island of the IGF2 gene in Bos taurus indicus cumulus cells during in vitro maturation

The aim of this study was to characterise the methylation pattern in a CpG island of the IGF2 gene in cumulus cells from 1-3 mm and a parts per thousand yenaEuro parts per thousand 8.0 mm follicles and to evaluate the effects of in vitro maturation on this pattern.Genomic DNA was treatment with sodium bisulphite. Nested PCR using bisulphite-treated DNA was performed, and DNA methylation patterns have been characterised.There were no differences in the methylation pattern among groups (P > 0.05). Cells of pre-IVM and post-IVM from small follicles showed methylation levels of 78.17 +/- 14.11 % and 82.93 +/- 5.86 %, respectively, and those from large follicles showed methylation levels of 81.81 +/- 10.40 % and 79.64 +/- 13.04 %, respectively. Evaluating only the effect of in vitro maturation, cells of pre-IVM and post-IVM COCs showed methylation levels of 80.17 +/- 12.01 % and 81.19 +/- 10.15 %.In conclusion, the methylation levels of the cumulus cells of all groups were higher than that expected from the imprinted pattern of somatic cells. As the cumulus cells from the pre-IVM follicles were not subjected to any in vitro manipulation, the hypermethylated pattern that was observed may be the actual physiological methylation pattern for this particular locus in these cells. Due the importance of DNA methylation in oogenesis, and to be a non-invasive method for determining oocyte quality, the identification of new epigenetic markers in cumulus cells has great potential to be used to support reproductive biotechniques in humans and other mammals.

Limitations of enamel microabrasion technique applied in a pediatric patient: case report

Enamel microabrasion is a non-invasive method that removes intrinsic and superficial defects from teeth aimed to improve dental esthetic with minimal loss of dental tissue. This case presentation describes the attempt for teeth color correction utilizing that conservative technique in a young girl whose upper central incisor presented an opaque white stain. Scanning electron microscopy (SEM) was conducted in order to illustrate the glasslike luster and a smooth texture of microabraded enamel surface. The correct diagnosis of defect is a difficult task, when consider this conservative approach.