Nonhuman primate breath volatile organic compounds associate with developmental programming and cardio-metabolic status.

Rodent and nonhuman primate studies indicate that developmental programming by reduced perinatal nutrition negatively impacts life course cardio-metabolic health. We have developed a baboon model in which we feed control mothers (CON) ad libitum while nutrient restricted mothers are fed 70% of ad libitum global feed in pregnancy and lactation. Offspring of nutrient restricted mothers are intrauterine growth restricted (IUGR) at term. By 3.5 years IUGR baboons showed signs of insulin resistance, indicating a pre-diabetic phenotype, in contrast to healthy CON offspring. We hypothesized that a novel breath analysis approach would provide markers of the altered cardio-metabolic state in a non-invasive manner. Here we assess whether exhaled breath volatile organic compounds (VOCs) collected from this unique cohort of juvenile baboons with documented cardio-metabolic dysfunction resulting from in utero programming can be detected from their breath signatures. Breath was collected from male and female CON and IUGR baboons at 4.8 ± 0.2 years (human equivalent ∼13 years). Breath VOCs were quantified using a two-dimensional gas chromatography mass spectrometer. Two-way ANOVA, on 76 biologically relevant VOCs identified 27 VOCs (p < 0.05) with altered abundances between groups (sex, birthweight, and sex x birthweight). The 27 VOCs included 2-pentanone, 2-octanone, 2,2,7,7-tetramethyloctane and 3-methyl-1-heptene, which have not previously been associated with cardio-metabolic disease. Unsupervised principal component analysis of these VOCs could discriminate the four clusters defining males, females, CON and IUGR. This study, which is the first to assess quantifiable breath signatures associated with cardio-metabolic programing for any model of IUGR, demonstrates the translational value of this unique model to identify metabolites of programmed cardio-metabolic dysfunction in breath signatures. Future studies are required to validate the translatability of these findings to humans.

Breath biomarkers of whole-body gamma irradiation in the Göttingen minipig.

There is widespread interest in the development of tools to estimate radiation exposures. Exhaled breath provides a novel matrix for assessing biomarkers that could be correlated with exposures. The use of exhaled breath for estimating radiation exposure is warranted, as studies have shown that external exposure to ionizing radiation causes oxidative stress that accelerates lipid peroxidation of polyunsaturated fatty acids, liberating alkanes and alkane metabolites that are excreted in the breath as volatile organic compounds (VOCs). As a proof of principle study, small groups (n = 4) of Göttingen minipigs were whole-body irradiated with gamma rays delivered by a 60Co source at absorbed doses of 0, 0.25, 0.5, 0.75, 1, 1.25, 2, and 4 Gy. Additional groups (n = 4) were treated with lipopolysaccharide (LPS) or granulocyte colony stimulating factor (G-CSF), with and without concurrent 60Co exposure, at an absorbed dose of 1 Gy. Breath and background air VOC samples were collected on days -3, -2, -1, 0 pre-irradiation, then at 0.25, 24, 48, 72, and 168 h post-irradiation. VOCs were analyzed by automated thermal desorption with two-dimensional gas chromatography and time-of-flight mass spectrometry (ATD GCxGC TOF MS). The results show significant changes in 58 breath VOCs post-irradiation, mainly consisting of methylated and other derivatives of alkanes, alkenes, and benzene. Using a multivariate combination of these VOCs, a radiation response function was constructed, which was significantly elevated at 15 min post irradiation and remained elevated throughout the study (to 168 h post irradiation). As a binary test of radiation absorbed doses ≥ 0.25 Gy, the radiation response function distinguished irradiated animals from shams (0 Gy) with 83-84% accuracy. A randomly derived radiation response function was robust: When half of the biomarkers were removed, accuracy was 75%. An optimally derived function with two biomarkers was 82% accurate. As a binary test of radiation absorbed doses ≥ 0.5 Gy, the radiation response function identified irradiated animals with an accuracy of 87% at 15 min post irradiation and 75.5% at 168 h post irradiation. Treatment with LPS and G-CSF did not affect the radiation response function. This proof-of-principle study supports the hypothesis that breath VOCs may be used for estimating radiation exposures. Further studies will be required to validate the sensitivity and specificity of these potential biomarkers.

Detection of an extended human volatome with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry.

BACKGROUND: Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GCxGC-TOF MS) has been proposed as a powerful new tool for multidimensional analysis of complex chemical mixtures. We investigated GCxGC-TOF MS as a new method for identifying volatile organic compounds (VOCs) in normal human breath. METHODS: Samples of alveolar breath VOCs and ambient room air VOC were collected with a breath collection apparatus (BCA) onto separate sorbent traps from 34 normal healthy volunteers (mean age = 40 yr, SD = 17 yr, male/female = 19/15). VOCs were separated on two serial capillary columns separated by a cryogenic modulator, and detected with TOF MS. The first and second dimension columns were non-polar and polar respectively. RESULTS: BCA collection combined with GC×GC-TOF MS analysis identified approximately 2000 different VOCs in samples of human breath, many of which have not been previously reported. The 50 VOCs with the highest alveolar gradients (abundance in breath minus abundance in ambient room air) mostly comprised benzene derivatives, acetone, methylated derivatives of alkanes, and isoprene. CONCLUSIONS: Collection and analysis of breath VOCs with the BCA-GC×GC-TOF MS system extended the size of the detectable human volatile metabolome, the volatome, by an order of magnitude compared to previous reports employing one-dimensional GC-MS. The size of the human volatome has been under-estimated in the past due to coelution of VOCs in one-dimensional GC analytical systems.

Detection of volatile biomarkers of therapeutic radiation in breath.

Breath testing could provide a rational tool for radiation biodosimetry because radiation causes distinct stress-producing molecular damage, notably an increased production of reactive oxygen species. The resulting oxidative stress accelerates lipid peroxidation of polyunsaturated fatty acids, liberating alkanes and alkane metabolites that are excreted in the breath as volatile organic compounds (VOCs). Breath tests were performed before and after radiation therapy over five days in 31 subjects receiving daily fractionated doses: 180-400 cGy d(-1) standard radiotherapy (n = 26), or 700-1200 cGy d(-1) high-dose stereotactic body radiotherapy (n = 5). Breath VOCs were assayed using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry. Multiple Monte Carlo simulations identified approximately 50 VOCs as greater-than-chance biomarkers of radiation on all five days of the study. A consistent subset of 15 VOCs was observed at all time points. A radiation response function was built by combining these biomarkers and the resulting dose-effect curve was significantly elevated at all exposures ⩾1.8 Gy. Cross-validated binary algorithms identified radiation exposures ⩾1.8 Gy with 99% accuracy, and ⩾5 Gy with 78% accuracy. In this proof of principal study of breath VOCs, we built a preliminary radiation response function based on 15 VOCs that appears to identify exposure to localized doses of 1.8 Gy and higher. VOC breath testing could provide a new tool for rapid and non-invasive radiation biodosimetry.

DISCO: distance and spectrum correlation optimization alignment for two-dimensional gas chromatography time-of-flight mass spectrometry-based metabolomics.

A novel peak alignment algorithm using a distance and spectrum correlation optimization (DISCO) method has been developed for two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC/TOF-MS)-based metabolomics. This algorithm uses the output of the instrument control software, ChromaTOF, as its input data. It detects and merges multiple peak entries of the same metabolite into one peak entry in each input peak list. After a z-score transformation of metabolite retention times, DISCO selects landmark peaks from all samples based on both two-dimensional retention times and mass spectrum similarity of fragment ions measured by Pearson's correlation coefficient. A local linear fitting method is employed in the original two-dimensional retention time space to correct retention time shifts. A progressive retention time map searching method is used to align metabolite peaks in all samples together based on optimization of the Euclidean distance and mass spectrum similarity. The effectiveness of the DISCO algorithm is demonstrated using data sets acquired under different experiment conditions and a spiked-in experiment.

At-column heating and a resistively heated, liquid-cooled thermal modulator for a low-resource bench-top GC x GC.

A transportable GC x GC instrument is under development for on-site applications that would benefit from the enhanced resolution and powers of detection, which can be achieved by this method. In the present study, a low-resource GC x GC instrument using an electrically heated and liquid-cooled single-stage thermal modulator that requires no cryogenic materials is evaluated. The instrument also uses at-column heating, thus eliminating the need for a convection oven to house the two columns. The stainless-steel modulator tube is coated with PDMS, which can be heated to 350 degrees C for sample injection into the second-dimension column. The modulator is cooled to -30 degrees C by a 100 mL/min flow of PEG by means of a commercial liquid chiller and a small recirculating pump. Resistive heating of the modulator tube is provided by a programmable power supply, which uses a voltage program that results in increasing modulator temperature during an analysis. This, together with more rapid cooling by the use of a liquid cooling medium, results in reduced solute breakthrough following each heating cycle as the modulator cools to a temperature where quantitative trapping resumes. As a result, modulated peak widths at half-height of less than 40 ms are observed. Design and performance details are presented along with chromatograms of gasoline and an essential oil sample.

Analysis of human breath samples with a multi-bed sorption trap and comprehensive two-dimensional gas chromatography (GCxGC).

A multi-bed sorption trap designed to quantitatively collect volatile organic compounds from large-volume vapor samples and inject them into a gas chromatograph is combined with a comprehensive two-dimensional gas chromatograph (GCxGC) for the analysis of organic compounds in human breath samples. The first-column effluent of the GCxGC is modulated by a single-stage, resistively-heated and air-cooled segment of 0.18-mm i.d. stainless steel column using the same stationary phase as the first column. Cooling gas is provided by a two-stage conventional refrigeration system, and thus no consumables other than carrier gas and electric power are required. The sorption trap uses four discreet beds, three containing different grades of graphitized carbon and one containing a carbon molecular sieve. The ordering of the beds in the trap tube is from the weakest to strongest adsorbent during sample collection. Breath samples are collected in gas sampling bags, and samples are passed through the trap at a flow rate of about 50 cm3/min. After sample collection, hydrogen carrier gas flow is initiated in the direction opposite to the sample collection flow, and the metal trap tube is resistively heated to inject a sample plug into the GCxGC. Performance data for the combined GCxGC/sorption-trap instrument is described, and human breath-sample chromatograms are presented.

Band acceleration device for enhanced selectivity with tandem-column gas chromatography.

An electrically heated and air cooled metal sheath surrounding the first 50 cm of the second column in a series-coupled, capillary-column ensemble of a non-polar and a polar column is used to obtain enhanced isothermal separation of component pairs that are separated by the first column in the ensemble but co-elute from the ensemble by virtue of the different selectivity of the two columns. As the first of the two components passes into the second column, a current pulsed through the metal sheath rapidly heats the first 50 cm of the second column thus accelerating the band for the first component. Ensemble retention-time shifts of several seconds are easily obtained. The device is then rapidly cooled to quiescent oven temperature by a flow of pressured air through the space between the metal sheath and the fused silica capillary column and an additional flow through a larger, co-axial plastic tube. Both heating and cooling require only a few seconds. If substantial cooling of the device occurs before the band for the second component enters the device, the band experiences less thermally-induced acceleration with the result that the separation of the two targeted components is enhanced in the ensemble chromatogram with no significant change in the pattern of peaks for the other mixture components. If the device is cooled to a temperature below oven temperature before the arrival of the band for the second component, this band will be slowed, and further enhancement of separation is achieved in the ensemble chromatogram. A band trajectory model, based on retention factor versus temperature data for the two components in the two columns, is used to predict peak separation and to aid in the selection of temperature-pulse initiation times.

Electrically heated, air-cooled thermal modulator and at-column heating for comprehensive two-dimensional gas chromatography.

An instrument for comprehensive two-dimensional gas chromatography (GCxGC) is described using an electrically heated and air-cooled thermal modulator requiring no cryogenic materials or compressed gas for modulator operation. In addition, at-column heating is used to eliminate the need for a convection oven and to greatly reduce the power requirements for column heating. The single-stage modulator is heated by current pulses from a dc power supply and cooled by a conventional two-stage refrigeration unit. The refrigeration unit, together with a heat exchanger and a recirculating pump, cools the modulator to about -30 degrees C. The modulator tube is silica-lined stainless steel with an internal film of dimethylpolysiloxane. The modulator tube is 0.18 mm i.d. x 8 cm in length. The modulator produces an injection plug width as small as 15 ms.