Towards optimizing the protocol for untargeted profiling of urine volatiles via gas chromatography-ion mobility spectrometry. A pilot study.

The analysis of volatile organic compounds (VOCs) present in various biological samples holds immense potential for non-invasive disease diagnostics and metabolic profiling. One of the biological fluids that are suitable for use in clinical practice is urine. Given the limited quantity of VOCs in the urine headspace, it's imperative to enhance their extraction into the gaseous phase and prevent any degradation of VOCs during the thawing process. The study aimed to test several key parameters (incubation time, temperature, and thawing) that can influence urine volatilome and monitor selected VOCs for their stability. The analysis in this study was performed using a BreathSpec® (G.A.S., Dortmund, Germany) device consisting of a gas chromatograph (GC) coupled with an ion mobility spectrometer (IMS). Testing three different temperatures and incubation times yielded a low number of VOCs (9 out of 34) that exhibited statistically significant differences. However, examining three thawing conditions revealed no VOCs with statistically significant changes. Thus, we conclude that urine composition remains relatively stable despite exposure to various thermal stresses.

NMR plasma metabolomics study of patients overcoming acute myocardial infarction: in the first 12 h after onset of chest pain with statistical discrimination towards metabolomic biomarkers.

Acute myocardial infarction (AMI) is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia is essential for the ischemic patient's prevention and treatment. Despite the great prevalence and incidence only a small number of studies utilize a metabolomic approach to describe AMI condition. Recent studies have shown the impact of metabolites on epigenetic changes, in these studies plasma metabolites were related to neurological outcome of the patients making metabolomic studies increasingly interesting. The aim of this study was to describe metabolomic response of an organism to ischemic stress through the changes in energetic metabolites and aminoacids in blood plasma in patients overcoming acute myocardial infarction. Blood plasma from patients in the first 12 h after onset of chest pain was collected and compared with volunteers without any history of ischemic diseases via NMR spectroscopy. Lowered plasma levels of pyruvate, alanine, glutamine and neurotransmitter precursors tyrosine and tryptophan were found. Further, we observed increased plasma levels of 3-hydroxybutyrate and acetoacetate in balance with decreased level of lipoproteins fraction, suggesting the ongoing ketonic state of an organism. Discriminatory analysis showed very promising performance where compounds: lipoproteins, alanine, pyruvate, glutamine, tryptophan and 3-hydroxybutyrate were of the highest discriminatory power with feasibility of successful statistical discrimination.

Methylation of MMP2, TIMP2, MMP9 and TIMP1 in abdominal aortic aneurysm.

OBJECTIVES: Abdominal aortic aneurysm (AAA) and its complications are among the most serious cardiovascular diseases and its occurrence has risen sharply in recent years. The aim of this pilot study is to explore the relationship between the methylation of matrix metalloproteinases and tissue inhibitors of the metalloproteinases genes' promoter region, and abdominal aortic aneurysm (AAA) through the detection of the methylation status of MMP2, TIMP2, TIMP1, and MMP9 genes in peripheral blood. METHODS: The study included 43 males with verified AAA (case group) and 34 healthy males (control group). The methylation status of the genes' promoter region was detected by methylation-specific polymerase chain reaction (MS-PCR). RESULTS: In adominal aortic aneurysm patients, the methylation ratio of MMP2 gene was positive in 9.3 % (4 cases), 2.3 % (1 case) had methylated TIMP2 gene, 7.0 % (3 cases) had methylated TIMP1 gene, while the methylation ratio of MMP9 gene was positive in 93.0 % (40 cases). In the control group, MMP2 gene was found to be methylated in 5.9 % (2 cases), 5.9 % of cases had methylated TIMP2 and TIMP1 genes (2 cases), and MMP9 gene was found to be methylated in 91.2 % (31 cases). CONCLUSION: In our pilot study, we found no association between DNA methylation of gelatinases and their tissue inhibitors, and the development of an abdominal aortic aneurysm (Tab. 2, Fig. 1, Ref. 27).

Investigation of association between genetic polymorphisms of MMP2, MMP8, MMP9 and TIMP2 and development of varicose veins in the Slovak Population - pilot study.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent metalloendopeptidases that degrades extracellular matrix (ECM) components. MMPs are associated with venous wall remodelling, proliferation, migration, phenotypic and functional transformation of vascular smooth muscle cells and ECM organization under the physiological and pathophysiological conditions. We investigated possible association of genetic promoter polymorphisms of MMP2 (rs243866), MMP8 (rs11225395), MMP9 (rs3918242) and TIMP2 (rs8179090) to varicose veins development in the Slovak population. Genomic DNA from 276 Slovak individuals (138 cases, 138 controls) was genotyped for selected SNPs (rs243866, rs11225395, rs3918242 and rs8179090) using the PCR-RFLP analysis. The data were analysed by chi-squared (chi2) test, logistic regression, and Mann-Whitney test. The risk of varicose veins development was evaluated in dominant, codominant and recessive genetic models. The statistical evaluation of selected polymorphisms in patients in all three genetic models has not shown a significant risk of varicose veins development. Our study has not shown the association between selected polymorphisms and increased risk of varicose veins development in Slovak population. More evidence with broaden sample size is needed.

The Role of Dysregulated MicroRNA Expression in Lung Cancer.

MicroRNAs (miRNAs) are a class of small single-stranded non-protein-coding RNAs that play important regulatory roles in many cellular processes including cell proliferation, differentiation, growth control, and apoptosis. They regulate gene expression on the posttranscriptional level by translational repression, mRNA cleavage, or mRNA degradation in various physiological and pathological processes. In addition, some miRNAs can function as oncogenes or tumor suppressors, so they can regulate several genes that play important roles in tumorigenesis. It was found that miRNAs are directly involved in many types of cancer, including lung cancer. Lung cancer is the leading cause of cancer mortality worldwide with a substantially low survival rate. In this work, we summarize recent findings related to miRNAs mechanisms of action and the role of their dysregulated expression in lung tumorigenesis. We describe the most important miRNAs involved in lung cancer development and targets of their activity. The understanding of the miRNA regulation in cancer may help better understand the molecular mechanisms of tumorigenesis and their importance in cancerous transformation.

Polymorphisms of DNA repair genes and lung cancer in chromium exposure.

Chromium is a well known carcinogen involved in the lung cancer development. Polymorphism of some of the DNA repair genes may be associated with elevated risk of cancerous transformation. In the present study, we investigated the polymorphisms of the following selected members of the base and nucleotide excision repair genes: XPC (Lys939Gln), XPD (Lys751Gln), XRCC1(Arg399Gln), and hOGG1(Ser326Ser), and the risk they present toward the development of lung cancer, with emphasis on the effect of chromium exposure. We analyzed 119 individuals; 50 patients exposed to chromium with diagnosed lung cancer and 69 healthy controls. Genotypes were determined by a PCR-RFLP method. We found a significantly increased risk of lung cancer development in XPD genotype Lys/Gln (OR=1.94; 95% CI=1.10-3.43; p=0.015) and in the gene combinations: XPD Lys/Gln+XPC Lys/Gln (OR=6.5; 95% CI=1.53-27.49; p=0.009) and XPD Lys/Gln+XPC Gln/Gln(OR=5.2; 95% CI=1.07-25.32; p=0.04). In conclusion, gene polymorphisms in the DNA repair genes may underscore the risk of lung cancer development in the chromium-exposed individuals.

DNA repair genes polymorphism and lung cancer risk with the emphasis to sex differences.

Polymorphisms in nucleotide and base excision repair genes are associated with the variability in the risk of developing lung cancer. In the present study, we investigated the polymorphisms of following selected DNA repair genes: XPC (Lys939Gln), XPD (Lys751Gln), hOGG1 (Ser326Cys) and XRCC1 (Arg399Gln), and the risks they present towards the development of lung cancer with the emphasis to gender differences within the Slovak population. We analyzed 761 individuals comprising 382 patients with diagnosed lung cancer and 379 healthy controls. Genotypes were determined by polymerase chain reaction/restriction fragment length polymorphism method. We found out statistically significant increased risk for lung cancer development between genders. Female carrying XPC Gln/Gln, XPC Lys/Gln+Gln/Gln and XRCC1 Arg/Gln, XRCC1 Arg/Gln+Gln/Gln genotypes had significantly increased risk of lung cancer corresponding to OR = 2.06; p = 0.04, OR = 1.66; p = 0.04 and OR = 1.62; p = 0.04, OR = 1.69; p = 0.02 respectively. In total, significantly increased risk of developing lung cancer was found in the following combinations of genotypes: XPD Lys/Gln+XPC Lys/Lys (OR = 1.62; p = 0.04), XRCC1 Gln/Gln+hOGG1 Ser/Ser (OR = 2.14; p = 0.02). After stratification for genders, the following combinations of genotype were found to be significant in male: XPD Lys/Gln+XPC Lys/Lys (OR = 1.87; p = 0.03), XRCC1 Arg/Gln+XPC Lys/Lys (OR = 4.52; p = 0.0007), XRCC1 Arg/Gln+XPC Lys/Gln (OR = 5.44; p < 0.0001). In female, different combinations of the following genotypes were found to be significant: XRCC1 Arg/Gln+hOGG1 Ser/Ser (OR = 1.98; p = 0.04), XRCC1 Gln/Gln+hOGG1 Ser/Ser (OR = 3.75; p = 0.02), XRCC1 Arg/Gln+XPC Lys/Gln (OR = 2.40; p = 0.04), XRCC1 Arg/Gln+XPC Gln/Gln (OR = 3.03; p = 0.04). We found out decreased cancer risk in genotype combinations between female patients and healthy controls: XPD Lys/Lys+XPC Lys/Gln (OR = 0.45; p = 0.02), XPD Lys/Gln+XPC Lys/Lys (OR = 0.32; p = 0.005), XPD Lys/Gln+XPC Lys/Gln (OR = 0.48; p = 0.02). Our results did not show any difference between pooled smokers and non-smokers in observed gene polymorphisms in the association to the lung cancer risk. However, gender stratification indicated the possible effect of heterozygous constitution of hOGG1 gene (Ser/Cys) on lung cancer risk in female non-smokers (OR = 0.20; p = 0.01) and heterozygous constitution of XPC gene (Lys/Gln) in male smokers (OR = 2.70; p = 0.01).