A new strategy based on real-time secondary electrospray ionization and high-resolution mass spectrometry to discriminate endogenous and exogenous compounds in exhaled breath

Breath is considered to be an easily accessible matrix, whose chemical composition relates to compounds present in blood. Therefore many metabolites are expected in exhaled breath, which may be used in the future for the development of diagnostic methods. In this article, a new strategy to discriminate between exhaled endogenous metabolites and exhaled exogenous contaminants by direct high-resolution mass spectrometry is introduced. The analysis of breath in real-time by secondary electrospray ionization mass spectrometry allows to interpret the origin of exhaled compounds. Exhaled metabolites that originate in the respiratory system show reproducible and significant patterns if plotted in real-time (>1 data point per second). An exhaled metabolite shows a signal that tends to rise at the end of a complete (forced) exhalation. In contrast, exogenous compounds, which may be present in room air, are gradually diluted by the air from the deeper lung and therefore show a trend of falling intensity. Signals found in breath by using this pattern recognition are linked to potential metabolites by comparison with online databases. In addition to this real-time approach, it is also shown how to combine this method with classical analytical methods in order to potentially identify unknown metabolites. Finally exhaled compounds following smoking a cigarette, chewing gum, or drinking coffee were investigated to underline the usefulness of this new approach.     

Electronic thrombocyte count from total blood]

Platelets were simultaneously counted in 52 persons by the conventional method of Cronkite and Brecher and with a new electronic cell volume analyser. The platelet counts estimated by the electronic analyser were 10% lower than that measured with the conventional method. This difference decreased to 1.5% when the same diluent was used for both methods. The described electronic counting method of platelets in whole blood is a simple procedure, which gives well reproducible and exact results.     

Desktop analysers: quality of results obtained by medical office personnel.

We carried out a study to evaluate the quality of results obtained by 14 nontechnical medical office personnel using desktop analysers. The instruments evaluated were the Reflotron analyser, the Seralyzer, the Vision analyser and the DT60 analyser. For precision studies low and high concentrations of control materials were used. For correlation studies the results obtained by the office personnel were compared with those obtained by a trained technologist. The coefficient of variation for the office personnel ranged from 3.0% to 8.1% with the Reflotron analyser, from 6.3% to 26.5% with the Seralyzer, from 1.0% to 4.1% with the Vision analyser and from 1.4% to 16.7% with the DT60 analyser. The correlation coefficient ranged from 0.970 to 0.997 with the Reflotron analyser, from 0.779 to 0.997 with the Seralyzer, from 0.975 to 0.998 with the Vision analyser and from 0.963 to 0.995 with the DT60 analyser. The proportion of results obtained by the office personnel that differed by more than 10% from those obtained by the technologist was 7% with the Reflotron analyser, 42% with the Seralyzer, 2% with the Vision analyser and 21% with the DT60 analyser. The instruments whose operation involves the least number of steps gave the most reliable results in the hands of medical office personnel.     

Neuroses and their pathogenesis

History and current state of the problem of experimental neuroses are reviewed in this article. The presented results of neurochemical, structural, and electroencephalographic investigations unravel earlier unknown aspects of the disease. Thus, circulatory cerebral hypoxia, as a principally new stage discovered for the first time in the development of an experimental neurosis allows the pathogenetically substantiated treatment with antioxidants to be applied in animal experiments and in clinical therapy of neurotic patients.     

A mathematical model for breath gas analysis of volatile organic compounds with special emphasis on acetone

Recommended standardized procedures for determining exhaled lower respiratory nitric oxide and nasal nitric oxide (NO) have been developed by task forces of the European Respiratory Society and the American Thoracic Society. These recommendations have paved the way for the measurement of nitric oxide to become a diagnostic tool for specific clinical applications. It would be desirable to develop similar guidelines for the sampling of other trace gases in exhaled breath, especially volatile organic compounds (VOCs) which may reflect ongoing metabolism. The concentrations of water-soluble, blood-borne substances in exhaled breath are influenced by: (i) breathing patterns affecting gas exchange in the conducting airways, (ii) the concentrations in the tracheo-bronchial lining fluid, (iii) the alveolar and systemic concentrations of the compound. The classical Farhi equation takes only the alveolar concentrations into account. Real-time measurements of acetone in end-tidal breath under an ergometer challenge show characteristics which cannot be explained within the Farhi setting. Here we develop a compartment model that reliably captures these profiles and is capable of relating breath to the systemic concentrations of acetone. By comparison with experimental data it is inferred that the major part of variability in breath acetone concentrations (e.g., in response to moderate exercise or altered breathing patterns) can be attributed to airway gas exchange, with minimal changes of the underlying blood and tissue concentrations. Moreover, the model illuminates the discrepancies between observed and theoretically predicted blood-breath ratios of acetone during resting conditions, i.e., in steady state. Particularly, the current formulation includes the classical Farhi and the Scheid series inhomogeneity model as special limiting cases and thus is expected to have general relevance for a wider range of blood-borne inert gases. The chief intention of the present modeling study is to provide mechanistic relationships for further investigating the exhalation kinetics of acetone and other water-soluble species. This quantitative approach is a first step towards new guidelines for breath gas analyses of volatile organic compounds, similar to those for nitric oxide.     

A rapid analytical technique for the determination of energy expenditure by the doubly labelled water method

The doubly labelled water method involves the administration of water enriched in 2H and 18O followed by determination of the turnover rates of these isotopes. Since 18O is eliminated from the body as both CO2 and water, while 2H leaves only as water, the difference between the two turnover rates provides a measure of CO2 production and hence energy expenditure. Isotopic analysis by conventional stable isotope ratio analysis (SIRA) is labour intensive and time consuming, as it requires off-line conversion of water samples to gases (H2 and CO2) followed by sequential analysis for each of the two isotopes using the mass spectrometer. Lack of suitable automated instrumentation with the ability to process large numbers of samples has prevented routine application of the method. We describe here an automated technique in which body water samples (urine, saliva, breath water or milk) are analysed simultaneously for 2H and 18O. The single bench system comprises two mass spectrometer analysers, one for measuring 2H from H2 gas, the other for measuring 18O from the water vapour (masses 18, 20). Both analysers share a common heated inlet system into which microlitre quantities of the body fluids are injected from an autosampler (102 samples). The water vapour flows both directly to one analyser for 18O measurement and into a uranium reduction furnace for conversion to H2, prior to 2H measurement by the second analyser. Both analysers also share vacuum and electronic components, enabling savings in both space and cost. In this paper we present results illustrating performance characteristics and procedures for routine application to human subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of α-amylase activity by Sequential Injection Analysis

Summary A Sequential Injection Analysis (SIA) system for monitoring -amylase activity is described. The SIA analyser is a further development of previously investigated Flow Injection Analysis (FIA) analyser. The analysis of -amylase activity is based on monitoring the decoloration of an iodine-starch complex. Performances of the SIA analyser have been compared with the FIA analyser. A good agreement has been obtained between the SIA measurements and the FIA measurements.     

基于生理征象变化的心理测试技术研究进展

本文从心理测试技术的起源和早期应用入手,介绍了心理状态与皮肤电、心率、脉搏、血压、呼吸、眼动等生理指标的关系,阐述了心理变化与生理征象变化之间的密切关系,揭示了心理测试技术的生理学依据.通过分阶段对萌芽期、近代、现代和当代心理测试仪器的设计思路和原理介绍,结合对典型的机械式、电子式和多元化测试仪器的功能,较为充分的展示了心理测试技术的研究进展及其在测试仪器改进上的体现.文章还介绍了基于脑电信号、事件相关电位、功能磁共振成像、核磁共振、皮肤温度、语音次声波等生理参数设计的正处于研发阶段的新型测试仪,并展望了心理测试技术发展和应用前景.     

Contractile responses of the airways smooth muscles in experimental bronchial asthma; the role of interleukin 5 in formation of hyper-responsiveness of bronchial tubes

By mehanographic method, contraktile responses of the airways smooth muscles in experimental bronchial asthma in porpoises intact and incubated with interleykin-5, were studied. Sensitization of ovalbumin animals results in development of hypersensitivity to inhalation of the fiber with external attributes of infringement of bronchial passableness such as cough and a shortness of breath. Morphologically the process is accompanied by destruction of epithelium of the bronchial tubes, development of an immune inflammation in a bronchial wall, and a hypertrophy of the muscular layer. In the sensitized animals, development of hyper-responsiveness of the airways smooth muscles to histamine was obvious as shown in a significant decrease of the threshold concentration and an increase in the maximal amplitude of reduction. Interleukin-5 was shown to strengthen this process. Responses to holinergic and R2-adrenerdic influences practically did not change. The data obtained corroborate the hypothesis of development of the interleukin-5-depending bronchial hyper-responsiveness in absence of eosinophile damage in the mucous membrane of the bronchial tubes.     

Analysis of fetal breathing in real time using a microprocessor

A system has been designed using an inexpensive microprocessor to analyze fetal breathing movements on a breath-by-breath basis in real time. An algorithm was developed which would recognize fetal breathing from the changes in tracheal pressure and which was capable of rejecting the artifactual changes caused by fetal or maternal movements. The tracheal pressure signal was digitized (at 51 samples/s), differentiated, and the start and peak of each breath was recognized from the zero-crossing points of the differentiated signal. Each breath was validated for size and shape according to a set of criteria incorporated into the breath recognition algorithm. On acceptance of the pressure change as a valid breath, the inspiratory time, breath amplitude, breath-to-breath interval, and inspiratory effort were calculated and stored in memory. The program was structured so that the microprocessor was able to accept new data and output summarizes of previous data concurrently. More than 95% of breaths in records contaminated with movement artifacts were recognized.     

Circadian Variation of the Human Metabolome Captured by Real-Time Breath Analysis

Circadian clocks play a significant role in the correct timing of physiological metabolism, and clock disruption might lead to pathological changes of metabolism. One interesting method to assess the current state of metabolism is metabolomics. Metabolomics tries to capture the entirety of small molecules, i.e. the building blocks of metabolism, in a given matrix, such as blood, saliva or urine. Using mass spectrometric approaches we and others have shown that a significant portion of the human metabolome in saliva and blood exhibits circadian modulation; independent of food intake or sleep/wake rhythms. Recent advances in mass spectrometry techniques have introduced completely non-invasive breathprinting; a method to instantaneously assess small metabolites in human breath. In this proof-of-principle study, we extend these findings about the impact of circadian clocks on metabolomics to exhaled breath. As previously established, our method allows for real-time analysis of a rich matrix during frequent non-invasive sampling. We sampled the breath of three healthy, non-smoking human volunteers in hourly intervals for 24 hours during total sleep deprivation, and found 111 features in the breath of all individuals, 36–49% of which showed significant circadian variation in at least one individual. Our data suggest that real-time mass spectrometric "breathprinting" has high potential to become a useful tool to understand circadian metabolism, and develop new biomarkers to easily and in real-time assess circadian clock phase and function in experimental and clinical settings.     

Simultaneous diffusion and convection in single breath lung washout

Two mathematical models of pulmonary single breath gas washout (one analytic, one numerical) are developed and their predictions compared with experimental data on human subjects. Weibel's 23 generation symmetric anatomical model is used as a guide to bronchial tree geometry. Experimental plots of nitrogen concentration versus volume expired, dead space versus breath holding time, and dead space versus tidal volume are compared with plots predicted by the models. Agreement is good. A plot of nitrogen concentration in the airways as predicted by the numerical model at different times during inhalation and exhalation of a single breath of oxygen is shown. Model predictions for changes in dead space with changes in washout gas and expiratory flow rate are discussed. Use of the analytic model for obtaining average values of the path length from mouth to alveoli in a given subject is discussed. To the extent of their agreement with experiment, the models provide a sound physical basis for the correlation of airway structure and function.     

Method for determination of methadone in exhaled breath collected from subjects undergoing methadone maintenance treatment

At present drugs of abuse testing using exhaled breath as specimen is only possible for alcohol. However, we recently discovered that using modern liquid chromatography–mass spectrometry technique amphetamine and methamphetamine is detectable in exhaled breath following intake in drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath from patients undergoing methadone maintenance treatment. Exhaled breath was collected from 13 patients after intake of the daily methadone dose. The compounds were trapped by filtering the air through a C18 modified silica surface. After elution of any trapped methadone the extract was analysed by a combined liquid chromatography–tandem mass spectrometry method. Recovery of trapped methadone from the filter surface was 96%, no significant matrix effect was observed, and the quantification using methadone-d3 as an internal standard was accurate (<10% bias) and precise (coefficient of variation 1.6–2.0%). Methadone was indisputably identified by means of the mass spectrometry technique in exhaled breath samples from all 13 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (±20%) and correct retention time. Excretion rates ranged from 0.39 to 78 ng/min. No methadone was detected in 10 control subjects. This finding confirms that breath testing is a new possibility for drugs of abuse testing. Collection of exhaled breath specimen is likely to be more convenient and safe as compared to other matrices presently in use.     

A single-breath technique with variable flow rate to characterize nitric oxide exchange dynamics in the lungs.

Current techniques to estimate nitric oxide (NO) production and elimination in the lungs are inherently nonspecific or are cumbersome to perform (multiple-breathing maneuvers). We present a new technique capable of estimating key flow-independent parameters characteristic of NO exchange in the lungs: 1) the steady-state alveolar concentration (C(alv,ss)), 2) the maximum flux of NO from the airways (J(NO,max)), and 3) the diffusing capacity of NO in the airways (D(NO,air)). Importantly, the parameters were estimated from a single experimental single-exhalation maneuver that consisted of a preexpiratory breath hold, followed by an exhalation in which the flow rate progressively decreased. The mean values for J(NO,max), D(NO,air), and C(alv,ss) do not depend on breath-hold time and range from 280-600 pl/s, 3.7-7.1 pl. s(-1). parts per billion (ppb)(-1), and 0.73-2.2 ppb, respectively, in two healthy human subjects. A priori estimates of the parameter confidence intervals demonstrate that a breath hold no longer than 20 s may be adequate and that J(NO,max) can be estimated with the smallest uncertainty and D(NO,air) with the largest, which is consistent with theoretical predictions. We conclude that our new technique can be used to characterize flow-independent NO exchange parameters from a single experimental single-exhalation breathing maneuver.     

The pathogenesis and control of Staphylococcus aureus-induced mastitis: study models in the mouse

The intramammary colonisation by Staphylococcus aureus provokes mastitis in the cow. Once established, the infection is difficult to eradicate with available therapies and may become chronic. The present article focuses on the use of the experimental mouse model of S. aureus-induced mastitis as a practical approach for the study of bovine mastitis. Results obtained regarding the pathogenesis of S. aureus and the development of new therapeutic approaches are discussed.     

Cardiac and respiratory responses in the climbing perchAnabas testudineus

Summary The heart rate ofAnabas is about 40 beats/min when breathing water and air. Tachycardia occurs as the fish takes an air breath and the rate declines as the breath holding continues but water breathing stops. Bradycardia is observed during this later phase. By the time the next air breath is taken, the heart rate has dropped to 15–30 beats/min.The heart rate of a fish breathing normoxic water but prevented from surfacing is about 42/min. Bradycardia occurs in hypoxic water under similar conditions. Tachycardia is observed in active fish when breathing hypercarbic water and air.On exposure to air bradycardia is found in resting fish but tachycardia occurs during activity. In air,Anabas usually repeats a breath when the heart rate drops to 15–25 beats/min. Tachycardia occurs immediately following an air breath. Heart rate then returns over a period of 3–6 mins to the normal resting level of 30 beats/min.The gas composition of the suprabranchial chambers has been measured and its relationship with the occurrence of an air breath, tachycardia and bradycardia is described.The experimental results are discussed in relation to the anatomy of the blood supply to the respiratory organs. The possible role of respiratory gases and pH in the control of breathing and perfusion is also discussed.We wish to thank the Nuffield Foundation for their financial support. We are also grateful to the Smithsonian Institution, Washington, D.C., and especially Dr. Stan Weitzman of the Division of Fishes, whose co-operation made this work possible.     

In-cell NMR spectroscopy.

The recent development of "in-cell NMR" techniques by two independent groups has demonstrated that NMR spectroscopy can be used to characterize the conformation and dynamics of biological macromolecules inside living cells. In this article, we describe different methods and discuss current and future applications as well as critical parameters of this new technique. We show experimental results, compare them with traditional in vitro experiments, and demonstrate that differences between the in vitro and the in vivo state of a macromolecule exist and can be detected and characterized.     

The automation of a manual amino acid analyser

The automation of a manual amino acid analyser is described. The modified analyser is fully automatic and will perform up to 19 analyses consecutively with a cycle time of 190 min. The modifications are simple and relatively inexpensive. The performance and reliability of the analyser are excellent.     

Trace gases in breath of healthy volunteers when fasting and after a protein-calorie meal: a preliminary study.

The selected ion flow tube technique was used to quantify in breath the trace gases acetone, ammonia, ethanol, isoprene, and methanol during single exhalations while fasting and in response to feeding. Six normal volunteers were fasted for 12 h, and, after baseline breath samples were obtained, were fed a liquid protein-calorie meal to provide 0.47 g/kg of protein (Fortisip). Further breath samples were obtained at 20, 40, and 60 min, and then hourly for a further 5 h. Breath acetone concentrations fell from a maximum during fasting, reaching their nadir between 4 and 5 h. Breath ammonia concentrations fell immediately to one-half their fasting levels before a steady increase to two or three times baseline values at 5 h. There was a brief increase in breath ethanol concentrations after feeding, reflecting detectable ethanol contamination of the food. Subsequently, breath ethanol levels remained low throughout the experimental protocol. Isoprene concentrations did not change significantly, whereas changes in methanol concentrations reflected those in the ambient air. This preliminary study indicates that the selected ion flow tube technique may be used to detect changes in the trace gases present in breath and define their concentrations in the fasting and replete state. Of particular interest is the biphasic response of the breath ammonia concentration after feeding.