Unravelling the origin of isoprene in the human body-a forty year Odyssey.

In the breath research community's search for volatile organic compounds that can act as non-invasive biomarkers for various diseases, hundreds of endogenous volatiles have been discovered. Whilst these systemic chemicals result from normal and abnormal metabolic activities or pathological disorders, to date very few are of any use for the development of clinical breath tests that could be used for disease diagnosis or to monitor therapeutic treatments. The reasons for this lack of application are manifold and complex, and these complications either limit or ultimately inhibit the analytical application of endogenous volatiles for use in the medical sciences. One such complication is a lack of knowledge on the biological origins of the endogenous volatiles. A major exception to this is isoprene. Since 1984, i.e. for 40 years, it has been generally accepted that the pathway to the production of human isoprene, and hence the origin of isoprene in exhaled breath, is through cholesterol biosynthesis via the mevalonate (MVA) pathway within the liver. However, various studies between 2001 and 2012 provide compelling evidence that human isoprene is produced in skeletal muscle tissue. A recent multi-omic investigation of genes and metabolites has revealed that this proposal is correct by showing that human isoprene predominantly results from muscular lipolytic cholesterol metabolism. Despite the overwhelming proof for a muscular pathway to isoprene production in the human body, breath research papers still reference the hepatic MVA pathway. The major aim of this perspective is to review the evidence that leads to a correct interpretation for the origins of human isoprene, so that the major pathway to human isoprene production is understood and appropriately disseminated. This is important, because an accurate attribution to the endogenous origins of isoprene is needed if exhaled isoprene levels are to be correctly interpreted and for assessing isoprene as a clinical biomarker.

A review on isoprene in human breath.

We summarize the history and review the literature on isoprene in exhaled breath and discuss the current evidence and models that describe its endogenous origin and consequence for understanding isoprene levels and their variations in exhaled breath.

Limonene in exhaled breath is elevated in hepatic encephalopathy.

Breath samples were taken from 31 patients with liver disease and 30 controls in a clinical setting and proton transfer reaction quadrupole mass spectrometry (PTR-Quad-MS) used to measure the concentration of volatile organic compounds (VOCs). All patients had cirrhosis of various etiologies, with some also suffering from hepatocellular cancer (HCC) and/or hepatic encephalopathy (HE). Breath limonene was higher in patients with No-HCC than with HCC, median (lower/upper quartile) 14.2 (7.2/60.1) versus 3.6 (2.0/13.7) and 1.5 (1.1/2.3) nmol mol-1 in controls. This may reflect disease severity, as those with No-HCC had significantly higher UKELD (United Kingdom model for End stage Liver Disease) scores. Patients with HE were categorized as having HE symptoms presently, having a history but no current symptoms and having neither history nor current symptoms. Breath limonene in these groups was median (lower/upper quartile) 46.0 (14.0/103), 4.2 (2.6/6.4) and 7.2 (2.0/19.1) nmol mol-1, respectively. The higher concentration of limonene in those with current symptoms of HE than with a history but no current symptoms cannot be explained by disease severity as their UKELD scores were not significantly different. Longitudinal data from two patients admitted to hospital with HE show a large intra-subject variation in breath limonene, median (range) 18 (10-44) and 42 (32-58) nmol mol-1.

Elimination characteristics of post-operative isoflurane levels in alveolar exhaled breath via PTR-MS analysis.

Isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether), C3H2ClF5O, is a commonly used inhalation anaesthetic. Using a proton transfer reaction mass spectrometer (PTR-MS) we have detected isoflurane in the breath of patients several weeks following major surgery. That isoflurane is detected in the breath of patients so long after being anaesthetised raises questions about when cognitive function has fully returned to a patient. Temporal profiles of isoflurane concentrations in breath are presented for five patients (F/M 3/2, mean age 50 years, min-max 36-58 years) who had undergone liver transplant surgery. In addition, results from a headspace analysis of isoflurane are presented so that the product ions resulting from the reactions of H3O+ with isoflurane in PTR-MS could be easily identified in the absence of the complex chemical environment of breath. Six product ions were identified. In order of increasing m/z (using the 35Cl isotope where appropriate) these are [Formula: see text] (m/z 51), CHFCl+ (m/z 67), CF3CHCl+ (m/z 117), C3F4OCl+ (m/z 163), C3H2F4OCl+ (m/z 165), and C3F4OCl+ H2O (m/z 183). No protonated parent was detected. For the headspace study both clean air and CO2 enriched clean air (4% CO2) were used as buffer gases in the drift tube of the PTR-MS. The CO2 enriched air was used to determine if exhaled breath would affect the product ion branching ratios. Importantly no significant differences were observed, and therefore for isoflurane the product ion distributions determined in a normal air mixture can be used for breath analysis. Given that PTR-MS can be operated under different reduced electric fields (E/N), the dependence of the product ion branching percentages for isoflurane on E/N (96-138 Td) are reported.

Volatile Biomarkers in Breath Associated With Liver Cirrhosis - Comparisons of Pre- and Post-liver Transplant Breath Samples.

BACKGROUND: The burden of liver disease in the UK has risen dramatically and there is a need for improved diagnostics. AIMS: To determine which breath volatiles are associated with the cirrhotic liver and hence diagnostically useful. METHODS: A two-stage biomarker discovery procedure was used. Alveolar breath samples of 31 patients with cirrhosis and 30 healthy controls were mass spectrometrically analysed and compared (stage 1). 12 of these patients had their breath analysed after liver transplant (stage 2). Five patients were followed longitudinally as in-patients in the post-transplant period. RESULTS: Seven volatiles were elevated in the breath of patients versus controls. Of these, five showed statistically significant decrease post-transplant: limonene, methanol, 2-pentanone, 2-butanone and carbon disulfide. On an individual basis limonene has the best diagnostic capability (the area under a receiver operating characteristic curve (AUROC) is 0.91), but this is improved by combining methanol, 2-pentanone and limonene (AUROC curve 0.95). Following transplant, limonene shows wash-out characteristics. CONCLUSIONS: Limonene, methanol and 2-pentanone are breath markers for a cirrhotic liver. This study raises the potential to investigate these volatiles as markers for early-stage liver disease. By monitoring the wash-out of limonene following transplant, graft liver function can be non-invasively assessed.

Use of proton transfer reaction time-of-flight mass spectrometry for the analytical detection of illicit and controlled prescription drugs at room temperature via direct headspace sampling.

The first reported use of proton transfer reaction time-of-flight mass spectrometry (PTR-TOF-MS) for the detection of a range of illicit and prescribed drugs is presented here. We describe the capabilities of PTR-TOF-MS to detect the following commonly used narcotics-ecstasy (N-methyl-3,4-methylenedioxyamphetamine), morphine, codeine, cocaine and heroin-by the direct sampling of the headspace above small solid quantities (approximately 50 mg) of the drugs placed in glass vials at room temperature, i.e. with no heating of the sample and no pre-concentration. We demonstrate in this paper the ability to identify the drugs, both illicit and prescribed, using PTR-TOF-MS through the accurate m/z assignment of the protonated parent molecule to the second decimal place. We have also included in this study measurements with an impure sample of heroin, containing typical substances found in "street" heroin, to illustrate the use of the technology for more "real-world" samples. Therefore, in a real-world complex chemical environment, a high level of confidence can be placed on the detection of drugs. Although the protonated parent is observed for all drugs, the reactant channel leading to this species is not the only one observed and neither is it necessarily the most dominant. Details on the observed fragmentation behaviour are discussed and compared to electrospray ionisation MS(n) studies available in the literature.

Proton transfer reaction mass spectrometry for the sensitive and rapid real-time detection of solid high explosives in air and water.

Relying on recent developments in proton transfer reaction mass spectrometry (PTR-MS), we demonstrate here the capability of detecting solid explosives in air and in water in real time. Two different proton transfer reaction mass spectrometers have been used in this study. One is the PTR-TOF 8000, which has an enhanced mass resolution (m/Δm up to 8,000) and high sensitivity (~50 cps/ppbv). The second is the high-sensitivity PTR-MS, which has an improved limit of detection of about several hundreds of parts per quadrillion by volume and is coupled with a direct aqueous injection device. These instruments have been successfully used to identify and monitor the solid explosive 2,4,6-trinitrotoluene (TNT) by analysing on the one hand the headspace above small quantities of samples at room temperature and from trace quantities not visible to the naked eye placed on surfaces (also demonstrating the usefulness of a simple pre-concentration and thermal desorption technique) and by analysing on the other hand trace compounds in water down to a level of about 100 pptw. The ability to identify even minute amounts of threat compounds, such as explosives, particularly within a complex chemical environment, is vital to the fight against crime and terrorism and is of paramount importance for the appraisal of the fate and harmful effects of TNT at marine ammunition dumping sites and the detection of buried antipersonnel and antitank landmines.

Low-energy electron attachment to the dichlorodifluoromethane (CCl2F2) molecule.

Results from a joint experimental study of electron attachment to dichlorodifluoromethane (CCl(2)F(2)) molecules in the gas phase are reported. In a high resolution electron beam experiment involving two versions of the laser photoelectron attachment method, the relative cross section for formation of the dominant anion Cl(-) was measured over the energy range 0.001-1.8 eV at the gas temperature T(G) = 300 K. It exhibits cusp structure at thresholds for vibrational excitation of the nu(3)(a(1)) mode due to interaction with the attachment channels. With reference to the thermal attachment rate coefficient k(T = 300 K) = 2.2(8) x 10(-9) cm(3) s(-1) (fitted average from several data), a new highly resolved absolute attachment cross section for T(G) = 300 K was determined. Partial cross sections for formation of the anions Cl(-), Cl(2)(-), F(-), ClF(-), and CCl(2)F(-) were measured over the range 0-12 eV, using three different electron beam experiments of medium energy resolution. The dependence of the attachment rate coefficient k(T(e);T(G) = 300 K) on electron temperature T(e) was calculated over the range 50-15 000 K, based on a newly constructed total cross section for anion formation at T(G) = 300 K. R-matrix calculations for Cl(-) production have been carried out for comparison with the experimental data. The R-matrix results are in line with the main experimental observations and predict the dependence of the DEA cross section on the initial vibrational level nu(3)() and on the vibrational temperature. Furthermore, the cross section for vibrational excitation of the nu(3) mode has been computed.

A preliminary comparison of volatile organic compounds in the headspace of cultures of Staphylococcus aureus grown in nutrient, dextrose and brain heart bovine broths measured using a proton transfer reaction mass spectrometer.

The effect of different growth media on the types and intensities of volatile organic compounds (VOCs) emitted by Staphylococcus aureus has been investigated using a proton transfer reaction mass spectrometer (PTR-MS). The microbial culture was grown in three different broths (nutrient, dextrose and brain heart bovine). Sterile sampling flasks contained either actively growing S. aureus or uninoculated broth. The headspace VOCs in these flasks were sequentially sampled and analysed. From the mass spectra obtained we selected the product ions (resulting from emissions from the inoculated samples) to monitor changes in their intensities as a function of time. Whilst the VOCs emitted by S. aureus in each medium were observed to be the same, the time-dependent pattern of growth showed marked differences and intensities between the different growth media. We conclude that PTR-MS provides sufficient sensitivity for in vivo microbial diagnosis: this could lead to more rapid treatment of bloodstream infections, particularly crucial for the well-being of immunocompromised and intensive care patients.

Endogenous volatile organic compounds in breath and blood of healthy volunteers: examining breath analysis as a surrogate for blood measurements.

To investigate the premise that levels of endogenous volatile organic compounds (VOC) in breath reflect those in blood, the concentration of acetone and isoprene were measured in radial arterial blood, peripheral venous blood and breath samples from ten healthy volunteers. Coefficients of repeatability as a percentage of mean are less than 30% in breath but greater than 70% in blood. The volunteer-mean ratios of arterial to venous blood concentration are 1.4 (0.9-2.1) for acetone and 0.55 (0.3-1.0) for isoprene. Concentration in breath showed a significant inter-subject correlation with concentration in arterial blood (CAB) for acetone but not for isoprene. Arterial blood/breath ratios are 580 (280-1060) for acetone and 0.47 (0.22-0.77) for isoprene. The sample-mean blood/breath ratio was used to calculate an estimate of CAB and the standard deviation of this estimate was lower than that of arterial blood measured directly. For most subjects, estimated CAB was within uncertainty limits of the actual CAB. Owing to the poor repeatability of VOC concentrations from consecutive blood samples, and the capacitive effects of the lung, this study suggests that breath VOC measurements may provide a more consistent measure than blood measurements for investigating underlying physiological function or pathology within individuals.

Development of a protocol to measure volatile organic compounds in human breath: a comparison of rebreathing and on-line single exhalations using proton transfer reaction mass spectrometry.

Analysis of volatile organic compounds (VOCs) on human breath has great potential as a non-invasive diagnostic technique. It is, therefore, surprising that no single, standard procedure has evolved for breath sampling. Here we present a novel repeated-cycle isothermal rebreathing method, where one cycle comprises five rebreaths, which could be adopted for breath analysis of VOCs. For demonstration purposes, we present measurements of three common breath VOCs: isoprene, acetone and methanol. Their concentrations measured in breath are shown to increase with number of rebreaths until a plateau value is reached by at least 20 rebreaths. The average ratio of plateau concentration to single mixed expired breath concentration was found to be 1.92 +/- 0.57 for isoprene, 1.25 +/- 0.13 for acetone and 1.12 +/- 0.12 for methanol (mean +/- standard deviation). Measurements from on-line single exhalations are presented which demonstrate a positive slope in the time-dependent expirograms of isoprene and acetone. The slope of the isoprene expirogram is persistently linear and the end-expired concentration of isoprene is highly variable in the same subject depending on the duration of exhalation. End-expired values of acetone are not as sensitive to the length of exhalation, and are the same to within measurement uncertainty for any duration of exhalation for any subject. It is concluded that uncontrolled single on-line exhalations are not suitable for the reliable measurement of isoprene in the breath and that rebreathing can be the basis of an easily tolerated protocol for the reliable collection of breath samples.

Real-time breath monitoring of propofol and its volatile metabolites during surgery using a novel mass spectrometric technique: a feasibility study.

BACKGROUND: At present, there is no rapid method for determining the plasma concentration of i.v. anaesthetics. A solution might be the measurement of the anaesthetic concentration in expired breath and its relation to the plasma concentration. We used chemical ionization methods to determine whether an i.v. anaesthetic can be detected in the low concentrations (parts per billion by volume) in the expired breath of an anaesthetized patient. METHOD: Chemical ionization mass spectrometry can measure trace gases in air with high sensitivity without interference from major gases. We carried out a feasibility trial with a proton transfer reaction mass spectrometer (PTR-MS) to monitor the i.v. anaesthetic agent propofol and two of its metabolites in exhaled gas from an anaesthetic circuit. Exhaled gas was sampled via a 4 m long, unheated tube connected to the PTR-MS. RESULTS: Propofol and its metabolites were monitored in real time in the expired breath of patients undergoing surgery. CONCLUSION: Routine measurement of i.v. agents, analogous to that for volatile anaesthetic agents, may be possible.