Chemical signature of colorectal cancer: case-control study for profiling the breath print.

BACKGROUND: Effective screening for colorectal cancer can reduce mortality by early detection of tumours and colonic polyps. An altered pattern of volatile organic compounds (VOCs) in exhaled breath has been proposed as a potential non-invasive diagnostic tool for detection of cancer. The aim of this study was to evaluate the reliability of breath-testing for colorectal cancer screening and early diagnosis using an advanced breath sampler. METHODS: The exhaled breath of patients with colorectal cancer and non-cancer controls with negative findings on colonoscopy was collected using the ReCIVA® Breath Sampler. This portable device is able to capture the alveolar breath fraction without environmental contamination. VOCs were desorbed thermally and analysed by gas chromatography-mass spectrometry. The discriminatory ability of VOCs in detecting colorectal cancer was evaluated by receiver operating characteristic (ROC) curve analysis for each VOC, followed by cross-validation by the leave-one-out method, and by applying stepwise logistic regression analysis. RESULTS: The study included 83 patients with colorectal cancer and 90 non-cancer controls. Fourteen VOCs were found to have significant discriminatory ability in detecting patients with colorectal cancer. The model with the diagnosis of cancer versus no cancer resulted in a statistically significant likelihood of discrimination of 173·45 (P < 0·001), with an area under the ROC curve of 0·979. Cross-validation of the model resulted in a true predictive value for colorectal cancer of 93 per cent overall. Reliability of the breath analysis was maintained irrespective of cancer stage. CONCLUSION: This study demonstrated that analysis of exhaled VOCs can discriminate patients with colorectal cancer from those without. This finding may eventually lead to the creation of a smart online sensory device, capable of providing a binary answer (cancer/no cancer) and directing to further screening.

ANTECEDENTES: Un cribaje efectivo del cáncer colorrectal (colorectal cáncer, CRC) puede reducir la mortalidad mediante la detección precoz de cáncer/pólipos del colon. La identificación de un patrón de compuestos volátiles orgánicos (volatile organic compounds, VOCs) en el aire espirado se ha propuesto como un procedimiento potencial de diagnóstico no invasivo para la detección del cáncer. El objetivo de este estudio fue evaluar la factibilidad del test de la respiración para el cribaje del CRC y diagnóstico precoz empleando un equipo avanzado de muestreo del aliento. MÉTODOS: Se recogieron muestras de aire espirado de 83 pacientes con CRC y de 90 controles sin cáncer con colonoscopia negativa empleando el ReCIVA Breath Sampler©. Este equipo portátil es capaz de capturar la fracción de aire alveolar espirada ausente de contaminación ambiental. Los VOCs fueron aislados térmicamente y analizados mediante cromatografía de gases acoplada a espectrometría de masas. La capacidad discriminatoria de los VOCs para detectar pacientes con CCR se evaluó mediante un análisis de la curva ROC para cada VOC seguida de validación cruzada mediante el método ir eliminando paso a paso cada uno de los VOCs en un modelo de regresión logística. RESULTADOS: Se observó que 14 VOCs tenían habilidad discriminatoria significativa para la detección de pacientes con CRC. El modelo con el diagnóstico de cáncer versus no cáncer mostró una probabilidad estadísticamente significativa de 151,03 (P < 0,0001) con un área bajo la curva (area under the curve, AUC) de 0,963. En la validación cruzada del modelo se obtuvo un valor global predictivo verdadero para el CRC del 92,5%. La fiabilidad del análisis del aire espirado se mantuvo con independencia del estadio del cáncer. CONCLUSIÓN: Este estudio ha demostrado que el análisis de los VOCs en el aire espirado puede discriminar pacientes con CRC de pacientes sin cáncer. Este hallazgo podría ser de ayuda para diceñar un dispositivo sensorial inteligente en línea, capaz de proporcionar una respuesta binaria (cáncer/NO cáncer) y asimismo contribuir a la indicación de una futura colonoscopia.

A quasi non-destructive approach for amber geological provenance assessment based on head space solid-phase microextraction gas chromatography-mass spectrometry.

Head space (HS) solid-phase micro-extraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) was used to analyze the volatile fraction of ambers of different geological origin. In particular, Romanian (romanite) and Baltic (succinite) amber samples were studied. Both types of amber have nearly similar bulk chemical compositions and could probably reflect only some differences of paleobiological and/or diagenetic origin. The present study shows that amber head space fingerprint, obtained by SPME/GC-MS, can provide a simple and quasi non-destructive method capable of romanite/succinite differentiation. Among the numerous compounds present in the head space, a number of few informative variables could be selected that were able to differentiate the ambers as demonstrated by Principal Component and Cluster Analysis.

Solid phase microextraction--liquid chromatography (SPME-LC) determination of chloramphenicol in urine and environmental water samples.

A solid phase microextraction--liquid chromatography with ultraviolet detection (SPME-LC-UV) method for the determination of the antimicrobial agent chloramphenicol was developed. The performances of three commercially available fibers were compared; the Carbowax/TPR-100 was found to provide the most efficient extraction. All the aspects influencing the fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte were investigated. The method was eventually applied to the determination of the drug in both biological (urine) and environmental (tap and sea water) samples. The optimized procedure required a simple sample pretreatment, isocratic elution, and provided enough sensitivity for the analyte determination in the considered samples. The investigated linear ranges were 37-1000 ng/ml (urine), 0.1-10 ng/ml (tap water), 0.3-30 ng/ml (sea water). Within-day and between-days RSD% ranged between 5.5-6.2 and 8.7-9.0 (urine), 5.1-6.0 and 8.4-8.8 (tap water), 5.4-5.7 and 8.6-8.9 (sea water). Estimated LOD and LOQ were 37 and 95 ng/ml (urine), 0.1 and 0.3 ng/ml (tap water), 0.3 and 0.7 ng/ml (sea water).

Optimization of analytical and pre-analytical conditions for MALDI-TOF-MS human urine protein profiles.

Protein analysis in biological fluids, such as urine, by means of mass spectrometry (MS) still suffers for insufficient standardization in protocols for sample collection, storage and preparation. In this work, the influence of these variables on healthy donors human urine protein profiling performed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was studied. A screening of various urine sample pre-treatment procedures and different sample deposition approaches on the MALDI target was performed. The influence of urine samples storage time and temperature on spectral profiles was evaluated by means of principal component analysis (PCA). The whole optimized procedure was eventually applied to the MALDI-TOF-MS analysis of human urine samples taken from prostate cancer patients. The best results in terms of detected ions number and abundance in the MS spectra were obtained by using home-made microcolumns packed with hydrophilic-lipophilic balance (HLB) resin as sample pre-treatment method; this procedure was also less expensive and suitable for high throughput analyses. Afterwards, the spin coating approach for sample deposition on the MALDI target plate was optimized, obtaining homogenous and reproducible spots. Then, PCA indicated that low storage temperatures of acidified and centrifuged samples, together with short handling time, allowed to obtain reproducible profiles without artifacts contribution due to experimental conditions. Finally, interesting differences were found by comparing the MALDI-TOF-MS protein profiles of pooled urine samples of healthy donors and prostate cancer patients. The results showed that analytical and pre-analytical variables are crucial for the success of urine analysis, to obtain meaningful and reproducible data, even if the intra-patient variability is very difficult to avoid. It has been proven how pooled urine samples can be an interesting way to make easier the comparison between healthy and pathological samples and to individuate possible differences in the protein expression between the two sets of samples.

Determination of clenbuterol in human urine and serum by solid-phase microextraction coupled to liquid chromatography.

A solid-phase microextraction (SPME)-LC-UV method for the determination of the beta-adrenergic drug clenbuterol in human urine and serum samples was developed for the first time using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated fiber. The procedure required very simple sample pretreatments, isocratic elution, and provided highly selective extractions. All the aspects influencing fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte have been investigated. The linear ranges investigated in urine and serum were 10-500 and 5-500 ng/ml, respectively (that covers the typical clenbuterol concentration observed in biological fluids). Within-day and between-days R.S.D.% in urine ranged between 5.0-5.3 and 8.5-8.7, respectively, while in serum ranged between 5.5-5.9 and 8.7-9.1, respectively. Estimated LOD and LOQ were 9 and 32 ng/ml (spiked urine), respectively, and 5 and 24 ng/ml (spiked serum), respectively, well below the usual clenbuterol urinary and serum level.

Amino-bonded silica as stationary phase for liquid chromatographic determination of cyclopiazonic acid in fungal extracts.

A new high-performance liquid (HPLC) chromatographic method is described for cyclopiazonic acid (CPA) determination in fungal cultures on a propylamino-bonded stationary phase with a CH3CN/CH3COONH4 buffer as mobile phase. Retention of CPA on propylamino modified silica under acidic conditions (protonated amino groups and deprotonated CPA) is governed by a mixed ion-exchange-reversed-phase mechanism. In addition to non-polar (hydrophobic) interactions, polar interactions with the surface silanols are also possible and become important as the polarity of the mobile phase decreases. A detection limit of 25 pg of CPA standard is obtained that represents an improvement of more than two orders of magnitude compared to existing HPLC procedures. UV-detector response was linear to 200 ng of CPA. Fungal extracts can be analysed after a simple dilution step with UV diode array detection that provides peak identity/purity assessment. The suitability of the proposed method as a rapid confirmatory test to assess the toxigenic potential of different Aspergillus and Penicillium strains is demonstrated by the analysis of 54 fungal extracts.

Liquid chromatographic determination of urinary 5-methyl-2'-deoxycytidine and pseudouridine as potential biological markers for leukaemia.

A simple reversed-phase liquid chromatographic (LC) method for the determination of urinary 5-methyl-2'-deoxycytidine (m5dCyd), recently claimed (on the basis of an imuno-technique) to be a potential marker for leukaemia, has been developed. Sample pre-treatment is based on a microcolumn clean-up step with an average recovery of 79% and a RSD of 3%. Detection limit was 0.2 microg/ml which is about tenfold lower than levels previously measured by an ELISA method in urine of healthy individuals. The creatinine (Cre) excretion, necessary for normalising the m5dCyd excretion, was evaluated by ion-pair liquid chromatography which permitted the simultaneous determination of pseudouridine (psi), a modified nucleoside also potentially useful as a marker for leukaemia. The described LC procedures were applied to the analysis of urine samples from healthy individuals and leukaemia patients. While the urinary psi/Cre ratio was found significantly increased for leukaemia patients, the urinary m5dCyd levels in healthy individuals were below the detection limits and did not increase in presence of the malignant disease.

In vitro toxicity of N3-methyl-5'-deoxy-5-fluorouridine, a novel metabolite of doxifluridine: a bioanalytical investigation.

The cytotoxicity of N3-methyl-5'-deoxy-5-fluorouridine (N3-Me-5'-dFUR), a novel metabolite of the anticancer pro-drug 5'-deoxy-5-fluorouridine (5'-dFUR), has been evaluated by in vitro experiments with cultures of different cancer cell lines. The new metabolic product was found to be non-toxic in all the cell growth experiments performed. The absence of cytotoxicity could be explained by the observation that the metabolite was not recognized as a substrate by thymidine phosphorilase, the enzyme responsible for 5-fluorouracil (5-FU) release from doxifluridine, as ascertained by high-performance liquid chromatography/ultraviolet (HPLC-UV) analysis of the incubation mixture. The biomethylation process leading to N3-Me-5'-dFUR could be considered as a possible detoxification pathway, altering the drug bioavailability, in competition with 5'-dFUR cleavage to the active 5-FU.

Hypermethylation of replicating hepatic DNA following N-methyl-N-nitrosourea administration.

Changes in the degree of methylation of cytosine in DNA are considered to be mechanistically important in modulating gene expression. To gain a better understanding of the relationship(s) linking onco-proliferative processes and enzymatic DNA methylation, a study has been carried out on the hepatic DNA methylation pattern during DNA replication following partial hepatectomy (PH), mitogen treatment and N-methyl-N-nitrosourea (MNU) administration in rats. The following results were obtained: (i) DNA hypomethylation was seen during DNA synthesis, with each of the 3 stimuli, namely MNU administration, partial hepatectomy, and hepatomitogen treatment; (ii) the level of DNA hypomethylation was not quantificatively related to the extent of DNA replication as measured by incorporation of [3H]thymidine into hepatic DNA; (iii) MNU administration under conditions conducive to carcinogenic development, i.e. during the S phase of compensatory cell proliferation, caused hypermethylation of replicating hepatic DNA, as shown by HpaII and MspI restriction patterns.

Effect of MNU on the methylation pattern of hepatic DNA during compensatory cell proliferation.

We have used the initiation-promotion model of MNU-induced hepatocarcinogenesis to test the hypothesis that alteration of the methylation status of DNA cytosines could be involved in the initiation of carcinogenesis. In fact cell proliferation plays a fundamental role in the initiation of liver carcinogenesis and hepatocytes in the S phase are more sensitive towards MNU initiation than at other times in the cycle. The molecular mechanisms involved in these processes, however, are still poorly understood and it seemed of value to monitor the DNA methylation status in this system. The results obtained indicate that MNU hepatocarcinogenic action might consist also of the inhibition of DNA hypomethylation biologically associated with cell proliferation.

DNA hypomethylation during liver cell proliferation induced by a single dose of lead nitrate.

DNA hypomethylation has already been found in regenerating rat liver and in hepatic (pre)malignant lesions when compared to normal non dividing liver. Here we report that extensive hypomethylation of hepatic DNA occurs in mitogen-treated rat liver. This effect can be seen as early as 12 h after metal treatment and parallels the liver dimension changes. Thus the lowering of the DNA 5-methylcytosine content appears to be a properly characteristic of cellular proliferation, independently from being caused by partial hepatectomy, carcinogen treatments or mitogen administration.

Transitory DNA hypomethylation during liver cell proliferation induced by a single dose of lead nitrate.

In the present study we have examined the effect of a single dose of the mitogen lead nitrate (75 mumols/kg body wt) on the methylation status of hepatic DNA in male Wistar rats. It was found that extensive hypomethylation of hepatic DNA occurs in mitogen-treated rat liver. This effect could be seen as early as 12 h after metal treatment and parallels the changes in liver weight. Probing with the methylation-sensitive enzymes HpaII, MspI, and HaeIII confirmed HPLC analyses and showed that methylation at these sites was affected by lead treatment. DNA hypomethylation has already been found in regenerating rat liver and in hepatic (pre)malignant lesions when compared to normal nondividing liver. Thus the lowering of the DNA 5-methylcytosine content appears to be a property characteristic of cellular proliferation, regardless of whether it is caused by partial hepatectomy, carcinogen treatments, or mitogen administration.

Effect of chemical carcinogens and partial hepatectomy on "in vivo" (35S)methionine interaction with rat liver tRNA.

The effect of carcinogens given by a single or multiple injections on the extent of (35S)methionine interaction with hepatic tRNA was studied in normal and partially hepatectomized rats. Either partial hepatectomy or administration of ethionine (100 or 330 mg/kg body weight) and dimethylnitrosamine (120 mg/kg body weight) by multiple i.p. injections inhibited the (35S)methionine-tRNA interaction, while administration of hepatocarcinogenic chemicals plus PH resulted rather in a stimulation. Methylnitrosourea enhanced the extent of interaction when administered in a single dose (100 mg per kg body weight) 18 h after partial hepatectomy.