A Proof of Concept: Are Detection Dogs a Useful Tool to Verify Potential Biomarkers for Lung Cancer?

Early and reliable diagnostic test is essential for effective therapy of lung cancer. Volatile organic compounds that are characteristic for cancer could serve as valuable biomarkers in cancer diagnosis. Both trace analytical and detection dog approaches give some evidence for the existence of such biomarkers. In this proof of concept, study dogs and trace analysis were implemented in combination to gain more information concerning cancer biomarkers. Two dogs were trained to distinguish between absorbed breath samples of lung cancer patients and healthy persons and succeeded with correct identification of patients with 9/9 and 8/9 and correct negative indications from of 8/10 and 4/10 samples from healthy individuals. A recent observational study found that breath samples from lung cancer patients showed an increase in 1-butanol, 2-butanone, 2-pentanone, and hexanal. Synthetic air samples were therefore fortified with these compounds and adsorbed to a fleece. Tested against breath samples from healthy probands, on presentation to the dogs these synthetic samples provoked an indication in three out of four samples. We were able to demonstrate that a combination of the natural nose of a dog and a trace analytic technique can be a valuable concept in the search for cancer biomarkers.

Odor Perception by Dogs: Evaluating Two Training Approaches for Odor Learning of Sniffer Dogs.

In this study, a standardized experimental set-up with various combinations of herbs as odor sources was designed. Two training approaches for sniffer dogs were compared; first, training with a pure reference odor, and second, training with a variety of odor mixtures with the target odor as a common denominator. The ability of the dogs to identify the target odor in a new context was tested. Six different herbs (basil, St. John's wort, dandelion, marjoram, parsley, ribwort) were chosen to produce reference materials in various mixtures with (positive) and without (negative) chamomile as the target odor source. The dogs were trained to show 1 of 2 different behaviors, 1 for the positive, and 1 for the negative sample as a yes/no task. Tests were double blind with one sample presented at a time. In both training approaches, dogs were able to detect chamomile as the target odor in any presented mixture with an average sensitivity of 72% and a specificity of 84%. Dogs trained with odor mixture containing the target odor had more correct indications in the transfer task.

Comparison of volatile organic compounds from lung cancer patients and healthy controls-challenges and limitations of an observational study.

This paper outlines the design and performance of an observational study on the profiles of volatile organic compounds (VOCs) in the breath of 37 lung cancer patients and 23 healthy controls of similar age. The need to quantify each VOC considered as a potential disease marker on the basis of individual calibration is elaborated, and the quality control measures required to maintain reproducibility in breath sampling and subsequent instrumental trace VOC analysis using solid phase microextraction-gas chromatography-mass spectrometry over a study period of 14 months are described. Twenty-four VOCs were quantified on the basis of their previously suggested potential as cancer markers. The concentration of aromatic compounds in the breath was increased, as expected, in smokers, while lung cancer patients displayed significantly increased levels of oxygenated VOCs such as aldehydes, 2-butanone and 1-butanol. Although sets of selected oxygenated VOCs displayed sensitivities and specificities between 80% and 90% using linear discriminant analysis (LDA) with leave-one-out cross validation, the effective selectivity of the breath VOC approach with regard to cancer detection is clearly limited. Results are discussed against the background of the literature on volatile cancer marker investigations and the prospects of linking increased VOC levels in patients' breath with approaches that employ sniffer dogs. Experience from this study and the literature suggests that the currently available methodology is not able to use breath VOCs to reliably discriminate between cancer patients and healthy controls. Observational studies often tend to note significant differences in levels of certain oxygenated VOCs, but without the resolution required for practical application. Any step towards the exploitation of differences in VOC profiles for illness detection would have to solve current restrictions set by the low and variable VOC concentrations. Further challenges are the technical complexity of studies involving breath sampling and possibly the limited capability of current analytical procedures to detect unstable marker candidates.

Investigation of cell culture volatilomes using solid phase micro extraction: Options and pitfalls exemplified with adenocarcinoma cell lines.

Three strategies to sample volatile organic compounds (VOC) from lung cancer cell lines cultured in vitro were compared. Headspace solid phase microextraction was applied in situ to culture flasks and alternatively to subsamples of headspace gas or to nutrient solution subsamples followed by gas chromatography-mass spectrometry. The direct quantification of 55 VOC in the headspace of cell cultures was validated and is discussed with respect to reproducibility and system-related interferences. The role of the VOC background from culture media and usually employed polystyrene culture vessels is examined and was seen to invoke potentially misleading conclusions. The commercial A549 and two further adenocarcinoma cell lines displayed largely similar VOC profiles with distinct differences regarding certain individual substances. There is evidence for the inappropriateness of the standard cell culturing methods in the search for volatile cancer markers.

In vitro cultured lung cancer cells are not suitable for animal-based breath biomarker detection.

In vitro cultured lung cancer cell lines were investigated regarding the possible identification of volatile organic compounds as potential biomarkers. Gas samples from the headspace of pure culture medium and from the cultures of human lung adenocarcinoma cell lines A549 and Lu7466 were exposed to polypropylene fleece in order to absorb odour components. Sniffer dogs were trained with loaded fleeces of both cell lines, and honey bees were trained with fleeces exposed to A549. Afterwards, their ability to distinguish between cell-free culture medium odour and lung cancer cell odour was tested. Neither bees nor dogs were able to discriminate between odours from the cancer cell cultures and the pure culture medium. Solid phase micro extraction followed by gas chromatography with mass selective detection produced profiles of volatiles from the headspace offered to the animals. The profiles from the cell lines were largely similar; distinct differences were based on the decrease of volatile culture medium components due to the cells' metabolic activity. In summary, cultured lung cancer cell lines do not produce any biomarkers recognizable by animals or gas chromatographic analysis.

Degradation of the ethyl glucuronide content in hair by hydrogen peroxide and a non-destructive assay for oxidative hair treatment using infra-red spectroscopy.

The assessment of quantification results of the alcohol abuse marker ethyl glucuronide (EtG) in hair in comparison to the cut-off values for the drinking behavior may be complicated by cosmetic hair bleaching. Thus, the impact of increasing exposure to hydrogen peroxide on the EtG content of hair was investigated. Simultaneously, the change of absorbance in the range of 1000-1100 cm(-1) indicative for the oxidation of cystine was investigated non-destructively by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) using pulverized portions of the respective hair samples. Hair samples treated with hydrogen peroxide consistently displayed a significantly increased absorbance at 1040 cm(-1) associated with the formation of cysteic acid. The EtG content decreased significantly if the hair was treated with alkaline hydrogen peroxide as during cosmetic bleaching. It could be shown that ATR-FTIR is capable of detecting an exposure to hydrogen peroxide when still no brightening was visible and already before the EtG content deteriorated significantly. Thus, hair samples suspected of having been exposed to oxidative treatment may be checked non-destructively by a readily available technique. This assay is also possible retrospectively after EtG extraction and using archived samples.

Determination of ethyl glucuronide in hair: a rapid sample pretreatment involving simultaneous milling and extraction.

A combination of simultaneous milling and extraction known as micropulverized extraction was developed for the quantification of the alcohol marker ethyl glucuronide (EtG) in hair samples using a homogeneous reference material and a mixer mill. Best extraction results from 50 mg of hair were obtained with 2-mL plastic tubes containing two steel balls (∅ = 5 mm), 0.5 mL of water and with an oscillating frequency of 30 s(-1) over a period of 30 min. EtG was quantified employing a validated GC-MS procedure involving derivatization with pentafluoropropionic acid anhydride. This micropulverization procedure was compared with dry milling followed by separate aqueous extraction and with aqueous extraction after manual cutting to millimeter-size snippets. Micropulverization yielded 28.0 ± 1.70 pg/mg and was seen to be superior to manually cutting (23.0 ± 0.83 pg/mg) and equivalent to dry grinding (27.7 ± 1.71 pg/mg) with regard to completeness of EtG extraction. The option to process up to 20 samples simultaneously makes micropulverization especially valuable for the high throughput of urgent samples.

Stabilisation of groundwater samples for the quantification of organic trace pollutants.

The concentration of contaminants in groundwater samples can be decreased by degradation in the time course between field sampling and quantification in the laboratory, especially in samples from sites where degradation activity is enhanced by remediation measures. The sampling sites covered a variety of priority organic pollutants such as volatile aromatic and chlorinated compounds, phenols and petroleum hydrocarbons and different remediation strategies such as anaerobic and aerobic microbial in situ degradation, in situ chemical oxidation, and on-site purification with biological treatment. The stability of the contaminants' concentration was investigated over a time range of several hours without cooling in the autosampler of the analytical equipment (short term) and over several days of storage until analysis (long term). A number of stabilisation techniques suggested in international standards ISO 5667-3:2013 and ASTM D6517:2000 were compared both with regard to short term and long term stabilisation of the contaminants and their practicability for field sampling campaigns. Long term storage turned out to be problematic for most compound groups even under cooling. Short term stability was problematic also for volatiles such as benzenic aromates, naphthalene and volatile organic halogenated compounds to be analysed by headspace gas chromatography. Acidification (pH <2) was sufficient to prevent degradation of benzenic aromates, naphthalene, phenols and petrol hydrocarbons for up to seven days. The use of acids was not applicable to stabilise volatiles in waters rich in carbonates and sulphides due to stripping of the volatiles with the liberated gases. The addition of sodium azide was successfully used for stabilisation of volatile organic halogenated compounds.

Quantification of ethyl glucuronide in hair: effect of milling on extraction efficiency.

AIM: The objective of the study was to provide conclusive evidence for the effect of particle size reduction as by milling on the extractable content of ethyl glucuronide (EtG) of hair samples. METHODS: A number of real case hair samples and two pooled hair materials with EtG contents in the range of 10-30 pg/mg were systematically compared with regard to the extraction yield of EtG after cutting to 2-3 mm length and pulverization with a ball mill. After the respective treatment the samples were submitted to aqueous extraction followed by quantification of EtG using HPLC-MS/MS. RESULTS: It was unequivocally demonstrated that milling of hair samples prior to aqueous extraction significantly increases the extractable EtG content compared with cut hair. The effect ranged between 137 and 230% and was seen to occur regardless of the extent of pulverization. Cooling of samples was not necessary to prevent partial degradation of EtG during the grinding procedure. CONCLUSION: The options currently employed at choice in analytical practice (cutting or milling) were seen to significantly affect the extractable amount of EtG in hair. This is suspected to influence the degree of equivalence of quantification results obtained in different laboratories as well as their respective classification of a test subject's drinking behaviour on the basis of currently recommended cut-off values.

1-(4-Bromo-3,5,5,6,8,8-hexa-methyl-5,6,7,8-tetra-hydro-naphthalen-2-yl)ethan-1-one: a precursor for phase-I metabolite of AHTN.

The title compound, C18H25BrO, crystallized as a racemate with four independent mol-ecules in the asymmetric unit. In the crystal, three of these four mol-ecules are linked via C-Br⋯Br-C halogen bonds [Br⋯Br = 3.662 (2) and 3.652 (2) Å], forming dimers.

Conformational analysis of alternariol on the quantum level.

With the help of theoretical calculations we explain the phenomenon of nonplanarity of crystalline alternariol. We find out that the different orientations of the hydroxyl groups of alternariol influence its planarity and aromaticity and lead to different twists of the structure. The presence of the intramolecular hydrogen bond stabilizes the planar geometry while the loss of the bond results in a twist of over 14°. This effect is thought to be involved while cutting DNA strands by alternariol.

Isopropyl 2,3,4,6-tetra-O-acetyl-ß-d-glucopyran-oside.

The title compound, C(17)H(26)O(10), was formed by a Koenigs-Knorr reaction of 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide and propan-2-ol. The central ring adopts a chair conformation. The crystal does not contain any significant inter-molecular inter-actions.

n-Propyl 2,3,4,6-tetra-O-acetyl-ß-d-glucopyran-oside.

THE TITLE COMPOUND [SYSTEMATIC NAME: (2R,3R,4S,5R,6R)-2-(acet-oxy-meth-yl)-6-propoxytetra-hydro-2H-pyran-3,4,5-triyl triacetate], C(17)H(26)O(10), was formed by a Koenigs-Knorr reaction of 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide and n-propanol. The central ring adopts a chair conformation. The crystal does not contain any significant inter-actions such as hydrogen bonds.

N-(ß-Carb-oxy-eth-yl)-α-isoleucine.

The title compound, {2-[(2-carbamoyleth-yl)amino]-3-methyl-penta-noic acid}, C(9)H(18)N(2)O(3), is of inter-est with respect to its biological activity. It was formed during an addition reaction between acryl-amide and the amino acid isoleucine. The crystal structure is a three-dimensional network built up by inter-molecular N-H⋯O and O-H⋯N hydrogen bonds.

The homogeneity testing of EtG in hair reference materials: a high-throughput procedure using GC-NCI-MS.

The validation of a robust quantification procedure for EtG in hair using GC-NCI-MS is presented. Aqueous extraction is followed by complete lyophylization of the extract and derivatization with pentafluoropropionic anhydride (PFPA) under controlled temperature and duration. Clean-up of extracts was dispensable and standard single quadrupole MS displayed sufficient selectivity and sensitivity. The method displayed a wide linearity range and enabled LOD of 0.68 pg/mg, LOQ of 2.4 pg/mg, and precision below 8.12%. Since EtG was seen to display prolonged stability in the aqueous extracts and after derivatization with PFPA this straightforward procedure allows a routine throughput of large quantities of samples with little proneness to procedural scatter of results. The method was applied to demonstrate the homogeneity of two hair reference materials with mean EtG contents of 8.48 pg/mg and 22.0 pg/mg. Aside from the application in homogeneity studies of hair reference materials predominantly in the concentration range of 10-50 pg/mg the method was also designed for daily routine quantification of real-world sample with regard to drinking behavior assessment.

Hydrothermally carbonized plant materials: patterns of volatile organic compounds detected by gas chromatography.

The nature and concentrations of volatile organic compounds (VOCs) in chars generated by hydrothermal carbonization (HTC) is of concern considering their application as soil amendment. Therefore, the presence of VOCs in solid HTC products obtained from wheat straw, biogas digestate and four woody materials was investigated using headspace gas chromatography. A variety of potentially harmful benzenic, phenolic and furanic volatiles along with various aldehydes and ketones were identified in feedstock- and temperature-specific patterns. The total amount of VOCs observed after equilibration between headspace and char samples produced at 270°C ranged between 2000 and 16,000µg/g (0.2-1.6wt.%). Depending on feedstock 50-9000µg/g of benzenes and 300-1800µg/g of phenols were observed. Substances potentially harmful to soil ecology such as benzofurans (200-800µg/g) and p-cymene (up to 6000µg/g in pine wood char) exhibited concentrations that suggest restrained application of fresh hydrochar as soil amendment or for water purification.

Degradation and epimerization of ergot alkaloids after baking and in vitro digestion.

The degradation and epimerization of ergot alkaloids (EAs) in rye flour were investigated after baking cookies and subsequently subjecting them to an in vitro digestion model. Different steps of digestion were analyzed using salivary, gastric, and duodenal juices. The degradation and bidirectional conversion of the toxicologically relevant (R)-epimers and the biologically inactive (S)-epimers for seven pairs of EAs were determined by a HPLC method coupled with fluorescence detection. Baking cookies resulted in degradation of EAs (2-30 %) and a shift in the epimeric ratio toward the (S)-epimer for all EAs. The applied digestion model led to a selective toxification of ergotamine and ergosine, two ergotamine-type EAs. The initial percentage of the toxic (R)-epimer in relation to the total toxin content was considerably increased after digestion of cookies. Ergotamine and ergosine increased from 32 to 51 % and 35 to 55 %, respectively. In contrast, EAs of the ergotoxine type (ergocornine, α- and ß-ergocryptine, and ergocristine) showed an epimeric shift toward their biologically inactive (S)-epimers. Further experiments indicated that the selective epimerization of ergotamine EAs occurs in the duodenal juice only. These results demonstrate that toxification of EAs in the intestinal tract should be taken into consideration.

Ergotaminine.

The title compound {systematic name: (6aR,9S)-N-[(2R,5S,10aS,10bS)-5-benzyl-10b-hy-droxy-2-methyl-3,6-dioxoocta-hydro-8H-oxazolo[3,2-a]pyrrolo-[2,1-c]pyrazin-2-yl]-7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo[4,3-fg]quinoline-9-carboxamide}, C(33)H(35)N(5)O(5), was formed by an epimerization reaction of ergotamine. The non-aromatic ring (ring C of the ergoline skeleton) directly fused to the aromatic rings is nearly planar [maximum deviation = 0.317 (4) Å] and shows an envelope conformation, whereas ring D, involved in an intra-molecular N-H⋯N hydrogen bond exhibits a slightly distorted chair conformation. The structure displays chains running approximately parallel to the diagonal of bc plane that are formed through N-H⋯O hydrogen bonds.

Koenigs-Knorr reaction of fusel alcohols with methyl (1-bromo-2,3,4-tri-O-acetyl-α-D-glucopyranosid)uronate leading to the protected alkyl glucuronides-crystal structures and high resolution 1H and 13C NMR data.

Crystal structures and high resolution (1)H and (13)C NMR spectral data for methyl (alkyl 2,3,4-tri-O-acetyl-ß-D-glucopyranosid)uronates (alkyl=methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, i-butyl, n-pentyl, 2-methyl-1-butyl and 3-methyl-1-butyl) are presented.

Lysergol monohydrate.

IN THE TITLE COMPOUND [SYSTEMATIC NAME: (7-methyl-4,6,6a,7,8,9-hexa-hydro-indolo[4,3,2-fg]quinoline-9-yl)methanol monohydrate], C(16)H(18)N(2)O·H(2)O, the non-aromatic ring (ring C of the ergoline skeleton) directly fused to the aromatic rings is nearly planar, with a maximum deviation of 0.659 (3) Å, and shows an envelope conformation. In the crystal, hydrogen bonds between the lysergol and water mol-ecules contribute to the formation of layers parallel to (10[Formula: see text]).