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Partial least squares-discriminant analysis (PLS-DA) scores scatter plots obtained for the HS-SPME/GC-MS chromatogram data of (A) VOCs and (B) VCCs of culture media of the metastatic cell lines vs. non-metastatic cell lines of each RCC histological subtype. (a, c) metastatic ccRCC (Caki-1, red, n = 11) vs. non-metastatic ccRCC (769-P and 786-O, dark blue, n = 27) and (b, d) metastatic pRCC (ACHN, green, n = 12) vs. non-metastatic pRCC (Caki-2, orange, n = 12).
Source publication
The identification of noninvasive biomarkers able to detect renal cell carcinoma (RCC) at an early stage remains an unmet clinical need. The recognition that altered metabolism is a core hallmark of cancer boosted metabolomic studies focused in the search for cancer biomarkers. The present work aims to evaluate the performance of the volatile metab...
Contexts in source publication
Context 1
... investigate the volatile exometabolome signature of RCC cell lines with different metastatic potential, the same strategy was applied to compare the extracellular medium of metastatic and nonmetastatic RCC cell lines (Figure 4). A good separation was obtained for metastatic vs. non-metastatic ccRCC cells in VOCs analysis (Q 2 > 0.7; Figure 4a), whereas a poor separation (Q 2 = 0.211) was observed in VCCs ( Figure 4c). ...
Context 2
... investigate the volatile exometabolome signature of RCC cell lines with different metastatic potential, the same strategy was applied to compare the extracellular medium of metastatic and nonmetastatic RCC cell lines (Figure 4). A good separation was obtained for metastatic vs. non-metastatic ccRCC cells in VOCs analysis (Q 2 > 0.7; Figure 4a), whereas a poor separation (Q 2 = 0.211) was observed in VCCs ( Figure 4c). For pRCC, a good separation with high predictive power (Q 2 > 0.9) was observed in both analytical methodologies (Figure 4b,d). ...
Context 3
... investigate the volatile exometabolome signature of RCC cell lines with different metastatic potential, the same strategy was applied to compare the extracellular medium of metastatic and nonmetastatic RCC cell lines (Figure 4). A good separation was obtained for metastatic vs. non-metastatic ccRCC cells in VOCs analysis (Q 2 > 0.7; Figure 4a), whereas a poor separation (Q 2 = 0.211) was observed in VCCs ( Figure 4c). For pRCC, a good separation with high predictive power (Q 2 > 0.9) was observed in both analytical methodologies (Figure 4b,d). ...
Context 4
... good separation was obtained for metastatic vs. non-metastatic ccRCC cells in VOCs analysis (Q 2 > 0.7; Figure 4a), whereas a poor separation (Q 2 = 0.211) was observed in VCCs ( Figure 4c). For pRCC, a good separation with high predictive power (Q 2 > 0.9) was observed in both analytical methodologies (Figure 4b,d). ...
Citations
... The most important ions associated with soil salinity are Na + and Cl − due to their negative effects on plants and soil, although other cations and anions (Ca 2+ , Mg 2+ , SO 4 2− or HCO 3 − ) may also have an influence [46]. In this sense, soil salinity affects various aspects of plant growth and development by imposing osmotic stress, ion toxicity, oxidative stress, and nutrient deficit (limiting the uptake of macro-and micronutrients) [5,7,34,35]. ...
... After each time, the VOCs production of GPTR29 was determined by headspace solid phase microextraction (HS-SPME-GC-MS) according to the methodology previously developed with minimal modifications [4]. Briefly, VOCs present in 4 mL of culture medium were extracted by HS-SPME using a divinylbenzene/ Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber under continuous stirring (250 rpm) with an incubation time of 5 min and extraction time of 30 min at 45 °C. ...
Background
Soils affected by salinity are a recurring problem that is continually increasing due to the impact of climate change on weather conditions and ineffective agricultural management practices. The use of plant growth promoting (PGP) Bacteria can alleviate its effects. In this regard, the genus Rhizobium has demonstrated excellent PGP capabilities through various plant growth promotion mechanisms and may therefore be a promising biofortifier under saline conditions. However, little is known about the production of volatile organic compounds (VOCs) by bacteria of this genus and their effects on plant development. Here, we aim to characterize the volatilome (the set of volatile metabolites synthesized by an organism) of Rhizobium for the first time and to further investigate the direct and VOC-mediated interaction between a strain of this genus and lettuce, a crop severely affected by salinity, both under saline and non-saline conditions.
Results
In this study, it was shown that the use of Rhizobium sp. GPTR29 was able to increase the production of lettuce (Lactuca sativa L.) under normal and saline conditions. We analyzed the Rhizobium volatilome under non-saline (0 mM NaCl) and saline (100 mM NaCl) conditions by HS-SPME-GC‒MS and found a differential composition in response to salinity. We detected 20 different compounds, where 3-methyl-1-butanol, 2-methyl-1-butanol, and α-pinene were the backbone of the Rhizobium volatilome. Exposure to these compounds in bicameral plates under salt stress resulted in increases in plant development of 17.1%, 16.0% and 33.1% in aerial part size, number of leaves and root length, respectively. Under greenhouse conditions and salinity, the inoculation of Rhizobium sp. GPTR29 resulted in an increase of 17.8% and 27.4% in shoot fresh and dry weight, respectively. Phenolic compounds were analyzed by HPLC–DAD-MS, revealing an increase in total flavonoid content under salinity conditions (100 mM NaCl) and apigenin derivative, luteolin 7-O-glucoside and quercetin 3-O-glucuronide individually.
Conclusions
These results provide new avenues for the study of PGP mechanisms in this bacterial genus, such as VOCs and their effects on plant growth, which play an important role in mediating plant–microorganism interactions.
Graphical abstract
... For instance, fruity scented breath can indicate diabetes [5], typhus skin infection smells like fresh-baking bread [6], bladder infection (cystitis) by E. coli causes cloudy, foul-smelling urine [7], and so on. Several applications of volatilomics for early diagnose of disease been reported in recent times [8][9][10]. Also, volatilomics can be used to infer drug concentration in human body. ...
... To assess the exometabolome (extracellular metabolites present in culture medium) of cells when exposed to MnTnHex and/or cisplatin, sample collection and preparation for analysis were performed according to a previously described protocol [45]. Briefly, A549 and H975 cells were seeded in 6-well plates at a density of approximately 8 × 10 4 and 9 × 10 4 cells/well, respectively, and incubated for 24 h in a complete cell culture medium. ...
... The GC-MS analysis was focused on the profile of volatile carbonyl compounds (VCCs), which include several aldehydes and ketones that may be produced as a result of oxidative stress. The preparation of all samples and VCCs extraction was performed according to previously described protocols based on headspace solid-phase microextraction (HS-SPME) [45,46]. Briefly, frozen samples were thawed slowly at room temperature. ...
The manganese(III) porphyrin MnTnHex-2-PyP5+ (MnTnHex) is a potent superoxide dismutase mimic and modulator of redox-based transcriptional activity that has been studied in the context of different human disease models, including cancer. Nevertheless, for lung cancer, hardly any information is available. Thus, the present work aims to fill this gap and reports the effects of MnTnHex in non-small cell lung cancer (NSCLC) cells, more specifically, A549 and H1975 cells, in vitro. Both cell lines were initially characterized in terms of innate levels of catalase, glutathione peroxidase 1, and peroxiredoxins 1 and 2. To assess the effect of MnTnHex in NSCLC, alone or in combination with cisplatin, endpoints related to the cell viability, cell cycle distribution, cell motility, and characterization of the volatile carbonyl compounds (VCCs) generated in the extracellular medium (i.e., exometabolome) were addressed. The results show that MnTnHex as a single drug markedly reduced the viability of both NSCLC cell lines, with some IC50 values reaching sub-micromolar levels. This redox-active drug also altered the cell cycle distribution, induced cell death, and increased the cytotoxicity pattern of cisplatin. MnTnHex also reduced collective cell migration. Finally, the metabolomics study revealed an increase in the levels of a few VCCs associated with oxidative stress in MnTnHex-treated cells. Altogether these results suggest the therapeutic potential of MnTnHex to be further explored, either alone or in combination therapy with cisplatin, in NSCLC.
... SPME's flexibility frequently enables its use to perform extractions from living cells and clinical specimens to identify correlations in the volatilome and potential biomarkers of various diseases. One example of such an application can be seen in a series of studies wherein the SPME was used to search for potential biomarkers of renal cell carcinoma (RCC) [4][5][6]. At first, SPME was used to study the volatilomes in urine samples from patients with different subtypes of RCC, which were then compared to those of a healthy control group. ...
... From the initial 21 volatile organic compounds (VOCs) differentiating the two groups, the authors eventually selected two metabolites, which passed the further stages of validation [4]. The subsequent investigation of potential biomarkers followed two directions: (1) more in-depth characterization of the urinary volatilome of clear cell renal cell carcinoma (ccRCC), which is one of the most common subtypes of RCC [5]; and (2) the differentiation of five different RCC cell lines based on their VOCs and volatile carbonyl compounds (VCCs), followed by the identification of metabolites that can potentially be used to discriminate between RCCs with different histological subtypes and metastatic potential [6]. ...
Solid-phase microextraction (SPME) possesses unique features that allow it to be used in analyses that would not be possible with traditional sample-preparation methods. The simplicity of SPME protocols and extraction devices makes it a uniform platform for analyzing biological samples, either via the headspace or in direct immersion mode. Furthermore, flexible probe design enables SPME to be applied to target objects of different sizes, offering analysis on a scale ranging "from single cell to living organs". SPME microfibers are minimally invasive, which enables them to be applied for the spatial and temporal monitoring of target analytes or to assess changes in the entire metabolome or lipidome. Furthermore, SPME permits the capture of the elusive portion of the metabolome, thus complementing exhaustive methods that are biased towards highly abundant and stable species. Significantly, SPME can be interfaced with analytical instrumentation to create a rapid diagnostic tool. However, despite these advantages, SPME has some limitations that must be well-understood and addressed. This paper presents examples of up-to-date applications of SPME, challenges related to particular studies, and future perspectives regarding the application of SPME in biomedical analysis.
... The analysis of the solutions was performed according to previous works [5,23]. Calcium (Ca), potassium (K), sodium (Na), and manganese (Mn) were determined by flame atomic absorption spectrometry (FAAS) using a PerkinElmer (Waltham, MA, USA) AAnalyst 200 instrument. ...
... For the analysis of VOCs (aldehydes, alcohols, acids, ketones, esters, acids, monoterpenes, norisoprenoids, heterocyclics, hydrocarbons, phenols, and phenylpropenes), the HS-SPME with a 50/30 μ m divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) (Supelco Bellefonte, Pennsylvania, USA) was used as described in previous studies, with minor modifications [5,23]. Approximately 0.1 g of lyophilized hydroethanolic extract, aqueous infusion, and crude extract of P. avium stems, leaves, and flowers were transferred to a 10 mL glass vial with 2 mL of H2O and 0.43 g of NaCl (salting out effect) and stirred (500 rpm) for 5 min. ...
Large amounts of Prunus avium L. by-products result from sweet cherry production and processing. This work aimed to evaluate the mineral content and volatile profiling of the cherry stems, leaves, and flowers of the Saco cultivar collected from the Fundão region (Portugal). A total of 18 minerals were determined by ICP-MS, namely 8 essential and 10 non-essential elements. Phosphorus (P) was the most abundant mineral, while lithium (Li) was detected in trace amounts. Three different preparations were used in this work to determine volatiles: hydroethanolic extracts, crude extracts, and aqueous infusions. A total of 117 volatile compounds were identified using HS-SPME/GC-MS, distributed among different chemical classes: 31 aldehydes, 14 alcohols, 16 ketones, 30 esters, 4 acids, 4 monoterpenes, 3 norisoprenoids, 4 hydrocarbons, 7 heterocyclics, 1 lactone, 1 phenol, and 2 phenylpropenes. Benzaldehyde, 4-methyl-benzaldehyde, hexanal, lilac aldehyde, and 6-methyl-5-hepten-2-one were the major volatile compounds. Differences in the types of volatiles and their respective amounts in the different extracts were found. This is the first study that describes the mineral and volatile composition of Portuguese sweet cherry by-products, demonstrating that they could have great potential as nutraceutical ingredients and natural flavoring agents to be used in the pharmaceutical, cosmetic, and food industries.
... In an in vitro metabolomics study, ketones were found to increase significantly in the exometabolome of RCC cells compared to a non-tumor cell line [60]. ...
Urine metabolomics profiling has potential for non-invasive RCC staging, in addition to providing metabolic insights into disease progression. In this study, we utilized liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and machine learning (ML) for the discovery of urine metabolites associated with RCC progression. Two machine learning questions were posed in the study: Binary classification into early RCC (stage I and II) and advanced RCC stages (stage III and IV), and RCC tumor size estimation through regression analysis. A total of 82 RCC patients with known tumor size and metabolomic measurements were used for the regression task, and 70 RCC patients with complete tumor-nodes-metastasis (TNM) staging information were used for the classification tasks under ten-fold cross-validation conditions. A voting ensemble regression model consisting of elastic net, ridge, and support vector regressor predicted RCC tumor size with a R2 value of 0.58. A voting classifier model consisting of random forest, support vector machines, logistic regression, and adaptive boosting yielded an AUC of 0.96 and an accuracy of 87%. Some identified metabolites associated with renal cell carcinoma progression included 4-guanidinobutanoic acid, 7-aminomethyl-7-carbaguanine, 3-hydroxyanthranilic acid, lysyl-glycine, glycine, citrate, and pyruvate. Overall, we identified a urine metabolic phenotype associated with renal cell carcinoma stage, exploring the promise of a urine-based metabolomic assay for staging this disease.
... It known that an acid environment leads to a larger number of VOCs produced compared with a neutral or alkaline environment [6,38]. Despite this, there is a lack of consistency in the approaches described in the literature with either acid, alkali, and/or salts used as a sample treatment [2,8,16,31,[41][42][43][44][45][46]. ...
Headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) can be used to measure volatile organic compounds (VOCs) in human urine. However, there is no widely adopted standardised protocol for the preparation of urine samples for analysis resulting in an inability to compare studies reliably between laboratories. This paper investigated the effect of altering urine sample pH, volume, and vial size for optimising detection of VOCs when using HS-SPME-GC-MS. This is the first, direct comparison of H2SO4, HCl, and NaOH as treatment techniques prior to HS-SPME-GC-MS analysis. Altering urine sample pH indicates that H2SO4 is more effective at optimising detection of VOCs than HCl or NaOH. H2SO4 resulted in a significantly larger mean number of VOCs being identified per sample (on average, 33.5 VOCs to 24.3 in HCl or 12.2 in NaOH treated urine) and more unique VOCs, produced a more diverse range of classes of VOCs, and led to less HS-SPME-GC-MS degradation. We propose that adding 0.2 mL of 2.5 M H2SO4 to 1 mL of urine within a 10 mL headspace vial is the optimal sample preparation prior to HS-SPME-GC-MS analysis. We hope the use of our optimised method for urinary HS-SPME-GC-MS analysis will enhance our understanding of human disease and bolster metabolic biomarker identification.
Polycystic ovary syndrome (PCOS) as a common metabolic and endocrinological disorder can affect the metabolic profile in biological fluids. We studied the profile of blood volatile organic compounds (VOCs) in rats with PCOS and controls to identify potential specific biomarkers of blood VOCs in PCOS rats. For this purpose, 30 female adult Wistar rats were assigned to two groups: control and PCOS groups. PCOS model was induced using letrozole gavage (1 mg/kg) for 21 days. The rats in the control group received water of the same volume for 21 days. During treatment, a collection of vaginal smears was done every day for estrus cycle determination and weight was measured weekly. On the day after the last administration of letrozole, the rats were killed and their blood and ovaries were collected. Testosterone levels and histologic changes in ovaries were examined. Also, headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC–MS) analyzed the VOCs in the blood of PCOS and control rats. Multivariate and univariate statistical analyses were used to find the potential biomarkers for a rat model of PCOS. Weight gain, ovarian and vaginal pathological alteration, as well as hyperandrogenemia, confirmed the successful induction of the PCOS in rats. The results of blood VOC analysis showed that nine VOCs were significantly elevated and one VOC decreased in the PCOS group than the control group (P<0/05). The partial least-squares discriminant analysis (PLS-DA) and principal component analysis (PCA) showed good separation of VOCs between the PCOS rats and the control group. The 4-ethylphenol and capric (decanoic) acid were selected as the potential biomarkers for PCOS diagnosis in the blood of the PCOS rats. The blood of PCOS rats had a specific profile of VOCs, which could be detected by GC-MS analysis. These findings can pave the way for further studies towards developing a new screening method for PCOS detection and studying their pathology, based on VOC analysis.
