Quantitative analysis of trace gases of breath during exercise using the new SIFT–MS technique

Abstract

We show how the concentration of the breath gases ammonia, acetone, and isoprene vary with time during exercise using the new selected ion flow tube mass spectrometry (SIFT–MS) technique. The expired breath concentrations of ammonia, acetone and isoprene were observed within the range of 50–500, 100–1400 and 5–400 ppb, respectively. Increasing acetone levels were observed for most subjects during the exercise period. However, isoprene levels decreased with time during exercise. Older subjects showed higher levels of isoprene compared with younger subjects. The ammonia time profile with exercise showed both decreasing and increasing patterns for different subjects.     

Real time analysis of breath volatiles using SIFT-MS in cigarette smoking

The selected ion flow tube mass spectrometry (SIFT-MS) technique enables real time analysis of trace volatiles at ppb levels without preconcentration steps or chemical derivatization. Most previous studies of trace compounds on the breath were analyzed using gas chromatography where enhanced detection sensitivity was achieved by concentrating the breath using cryogenic or adsorption trapping techniques. In this paper, we have examined volatile organic substances, isoprene, acetone, ammonia and ethanol in breath before and after smoking a cigarette. It is interesting that isoprene levels increased in all the subjects after smoking one cigarette with a mean increase of 70%. The mean increase for acetone was found to be 22%. In contrast to isoprene, a decreasing ethanol level was observed in all the subjects except one with the negative mean decrease of 28%. Further SIFT-MS studies also have high-lighted some organic substances produced even by unburned cigarettes, US and New Zealand products. Certain US brands have shown much higher levels of volatile species than cigarettes produced in New Zealand.     

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.     

Metabolic monitoring and assessment of anaerobic threshold by means of breath biomarkers

Volatile breath constituents such as acetone and ammonia have been linked to dextrose, fat, and protein metabolism. Non-invasive breath analysis, therefore, may be used for metabolic monitoring, identification of fuel sources actually used for energy production and determination of the anaerobic threshold (AT). This study was intended to assess correlations between exhaled volatile organic compound (VOC) concentrations, metabolism, and physiological parameters. In addition, we tried to find out whether AT could be determined by means of non-invasive analysis of VOCs in breath. Exhaled concentrations of acetone, ammonia, and isoprene were determined in 21 healthy volunteers under controlled ergometric exercise by means of continuous real time Proton Transfer Reaction Mass Spectrometry (PTR-MS). In parallel, spiro-ergometric parameters ( $ {\dot{\text{V}}} $ O₂, $ {\dot{\text{V}}} $ CO₂, respiratory rate and minute ventilation) and hemodynamic data such as heart rate were recorded. AT was determined from serum lactate, by means of respiratory exchange rate and by means of exhaled acetone concentrations. Exhaled acetone concentrations mirrored exercise induced changes of dextrose metabolism and lipolysis. Bland?CAltman statistics showed good agreement between lactate threshold, respiratory compensation point (RCP), and determination of AT by means of exhaled acetone. Exhaled ammonia concentration seemed to be linked to protein metabolism and changes of pH under exercise. Isoprene concentrations showed a close correlation to cardiac output and minute ventilation. Breath biomarkers represent a promising alternative for metabolic monitoring under exercise as they can be determined non-invasively and continuously. In addition, these markers may add complementary information on biochemistry, energy production and fuel consumption.     

Human breath isoprene and its relation to blood cholesterol levels: new measurements and modeling.

Numerous publications have described measurements of breath isoprene in humans, and there has been a hope that breath isoprene analyses could be a noninvasive diagnostic tool to assess blood cholesterol levels or cholesterol synthesis rate. However, significant analytic problems in breath isoprene analysis and variability in isoprene levels with age, exercise, diet, etc., have limited the usefulness of these measurements. Here, we have applied proton transfer reaction-mass spectrometry to this problem, allowing on-line detection of breath isoprene. We show that breath isoprene concentration increases within a few seconds after exercise is started as a result of a rapid increase in heart rate and then reaches a lower steady state when breath rate stabilizes. Additional experiments demonstrated that increases in heart rate associated with standing after reclining or sleeping are associated with increased breath isoprene concentrations. An isoprene gas-exchange model was developed and shows excellent fit to breath isoprene levels measured during exercise. In a preliminary experiment, we demonstrated that atorvastatin therapy leads to a decrease in serum cholesterol and low-density-lipoprotein levels and a parallel decrease in breath isoprene levels. This work suggests that there is constant endogenous production of isoprene during the day and night and reaffirms the possibility that breath isoprene can be a noninvasive marker of cholesterologenesis if care is taken to measure breath isoprene under standard conditions at constant heart rate.     

Breath isoprene: muscle dystrophy patients support the concept of a pool of isoprene in the periphery of the human body

Breath isoprene accounts for most of the hydrocarbon removal via exhalation and is thought to serve as a non-invasive indicator for assaying several metabolic effects in the human body. The primary objective of this paper is to introduce a novel working hypothesis with respect to the endogenous source of this compound in humans: the idea that muscle tissue acts as an extrahepatic production site of substantial amounts of isoprene. This new perspective has its roots in quantitative modeling studies of breath isoprene dynamics under exercise conditions and is further investigated here by presenting pilot data from a small cohort of late stage Duchenne muscle dystrophy patients (median age 21, 4 male, 1 female). For these prototypic test subjects isoprene concentrations in end-tidal breath and peripheral venous blood range between 0.09-0.47 and 0.11-0.72 nmol/l, respectively, amounting to a reduction by a factor of 8 and more as compared to established nominal levels in normal healthy adults. While it remains unclear whether isoprene can be ascribed a direct physiological mechanism of action, some indications are given as to why isoprene production might have evolved in muscle.     

Influence of dynamic hand-grip exercise on acetone in gas emanating from human skin

This study investigated the effects of dynamic hand-grip exercise on skin-gas acetone concentration. The subjects for this experiment were seven healthy males. In the first experiment, to ascertain the reproducibility of the results for the skin-gas acetone concentration test, the skin gas was collected four times from one subject. In the second experiment, all subjects performed three different types of exercise (Exercises I-III) for a duration of 60 s. Exercise I was performed at 10 kg with one contraction every 3 s. Exercise II was 30 kg with one contraction every 3 s. Exercise III was 10 kg with one contraction per second. Acetone concentration was analyzed by gas chromatography. In the first experiment, reasonable reproducibility was obtained in measurements of skin-gas acetone concentration during the hand-grip exercise. In the second experiment, acetone concentration in skin gas during hand-grip exercise II was significantly higher than the basal level. Although skin-gas acetone levels increased in all subjects during exercises I and III, a significant difference was not found. No significant difference was found in skin-gas acetone concentration during dynamic hand-grip exercise among exercises I, II, and III. This study confirmed that skin-gas acetone levels increase during dynamic hand-grip exercise.     

Impact of inspired substance concentrations on the results of breath analysis in mechanically ventilated patients.

A well-defined relationship has to exist between substance concentrations in blood and in breath if blood-borne volatile organic compounds (VOCs) are to be used as breath markers of disease or health. In this study, the impact of inspired substances on this relationship was investigated systematically. VOCs were determined in inspired and expired air and in arterial and mixed venous blood of 46 mechanically ventilated patients by means of SPME, GC/MS. Mean inspired concentrations were 25% of expired concentrations for pentane, 7.5% for acetone, 0.7% for isoprene and 0.4% for isoflurane. Only if inspired concentrations were <5% did substance disappearance rates from blood and exhalation rates correlate well. Exhaled substance concentrations depended on venous and inspired concentrations. Patients with sepsis had higher n-pentane and lower acetone concentrations in mixed venous blood than patients without sepsis (2.27 (0.37-8.70) versus 0.65 (0.33-1.48) nmol L-1 and 69 (22-99) versus 18 (6.7-56) micromol L-1). n-Pentane and acetone concentrations in breath showed no differences between the patient groups, regardless whether or not expired concentrations were corrected for inspired concentrations. In mechanically ventilated patients, concentration profiles of volatile substances in breath may considerably deviate from profiles in blood depending on the relative amount of inspired concentrations. A simple correction for inspired substance concentrations was not possible. Hence, substances having inspired concentrations>5% of expired concentrations should not be used as breath markers in these patients without knowledge of concentrations in blood and breath.     

Inhaled salbutamol decreases blood ammonia levels during exercise in normal subjects

The present study examines the effect of salbutamol, a beta2-adrenoreceptor agonist, on blood ammonia levels during an incremental cycle exercise test in healthy non-asthmatic subjects. Blood ammonia levels were lower after inhalation of 400 mcg of salbutamol than after placebo during submaximal exercise: 33+/-2 micromol x l(-1) v.s. 48+/-9 micromol x l(-1) at 220 W and 39+/-2 micromol x l(-1) v.s. 50+/-4 micromol x l(-1) at 260 W. At peak exercise there were no significant differences. The results suggest that beta2-adrenoreceptors are involved in the regulation of blood ammonia during exercise.     

Energy metabolism of medium-chain triglycerides versus carbohydrates during exercise

Medium-chain triglycerides (MCT) are known to be rapidly digested and oxidized. Their potential value as a source of dietary energy during exercise was compared with that of maltodextrins (MD). Twelve subjects exercised for 1 h on a bicycle ergometer (60% VO2 max), 1 h after the test meal (1MJ). The metabolism of MCT was followed using 1-13C-octanoate (Oc) as tracer and U-13C-glucose (G) was added to the 13C-naturally enriched MD. After MCT ingestion no insulin peak was observed with some accumulation of ketone bodies (KB), blood levels not exceeding 1 mM. Total losses of KB during exercise in urine, sweat and as breath acetone were small (less than 0.2 mmol X h-1). Hence, the influence of KB loss and storage on gas exchange data was negligible. The partition of fat and carbohydrate utilization during exercise as obtained by indirect calorimetry was practically the same after the MCT and the CHO meals. Oxidation over the 2-h period was 30% of dose for Oc and 45% for G. Glycogen decrements in the Vastus lateralis muscle were equal. It appears that with normal carbohydrate stores, a single meal of MCT or CHO did not alter the contribution of carbohydrates during 1 h of high submaximal exercise. The moderate ketonemia after MCT, despite substantial oxidation of this fat, led to no difference in muscle glycogen sparing between the diets.     

Exercise and recovery ventilatory and VO2 responses of patients with McArdle's disease

This study was designed to determine whether patients with McArdle's disease, who do not increase their blood lactate levels during and after maximal exercise, have a slow "lactacid" component to their recovery O2 consumption (VO2) response after high-intensity exercise. VO2 was measured breath by breath during 6 min of rest before exercise, a progressive maximal cycle ergometer test, and 15 min of recovery in five McArdle's patients, six age-matched control subjects, and six maximal O2 consumption- (VO2 max) matched control subjects. The McArdle's patients' ventilatory threshold occurred at the same relative exercise intensity [71 +/- 7% (SD) VO2max] as in the control groups (60 +/- 13 and 70 +/- 10% VO2max) despite no increase and a 20% decrease in the McArdle's patients' arterialized blood lactate and H+ levels, respectively. The recovery VO2 responses of all three groups were better fit by a two-, than a one-, component exponential model, and the parameters of the slow component of the recovery VO2 response were the same in the three groups. The presence of the same slow component of the recovery VO2 response in the McArdle's patients and the control subjects, despite the lack of an increase in blood lactate or H+ levels during maximal exercise and recovery in the patients, provides evidence that this portion of the recovery VO2 response is not the result of a lactacid mechanism. In addition, it appears that the hyperventilation that accompanies high-intensity exercise may be the result of some mechanism other than acidosis or lung CO2 flux.     

Ventilatory response to incremental and constant-workload exercise in the presence of a thoracic restriction.

In the presence of an externally applied thoracic restriction, conflicting ventilatory responses to exercise have been reported, which could be accounted for by differences in exercise protocol. Seven male subjects performed two incremental and two constant-workload ergometer tests either unrestricted or in the presence of an inelastic corset. Ventilatory variables and arterial estimates of PCO(2) were obtained breath by breath. Subjects hyperventilated in the presence of restriction during the constant-workload test (38.4 +/- 3.0 vs. 32.8 +/- 3.0 l/min for the average of the last 3 min of exercise, P < 0.05), whereas, at an equivalent workload during the incremental test, ventilation was similar to unrestricted values (unrestricted = 26.3 +/- 1.6 vs. restricted = 27.9 +/- 2.3 l/min, P = 0.36). We used a first-order linear model to describe the effects of change in workload on minute ventilation (24). When the time constants and minute ventilation values measured during unrestricted and restricted constant-workload exercise were used to predict the ventilatory response to the respective incremental exercise tests, no significant difference was observed. This suggests that hyperventilation is not seen in the restricted incremental test because the temporal dynamics of the ventilatory response are altered.     

Effects of postexercise supplementation of chicken essence on the elimination of exercise-induced plasma lactate and ammonia

We investigated the effects of chicken essence (CE) supplementation on exercise-induced changes of lactate and ammonia during recovery. In this randomized, double blind, crossover study, twelve healthy subjects performed a single bout of exercise to exhaustion, and then consumed either a placebo or CE within 5-min of the exercise cessation. Blood samples were collected before exercise, at exhaustion (0 minute), and 20, 40, 60, and 120 minutes, respectively during the recovery period. There were no differences in plasma glucose, creatine kinase, or heart rate responses between treatments. The exercise exhaustion significantly increased the levels of lactate and ammonia, and both measured values gradually declined during the recovery period. Ammonia levels at 40, 60, and 120 min. of the recovery period were observed lower significantly in the CE group, as compared to those in the placebo group. Additionally, lactate concentrations at 60 and 120 min were lower in the CE group, as compared to those in the placebo group. In conclusion, the main finding of this study was that CE supplementation after exercise reduces plasma lactate and ammonia levels. The results indicated that CE supplementation after an exhaustive exercise could enhance physiological recovery in humans.     

Pulmonary clearance of 99mTc-DTPA: influence of background activity

To study the effects of circulatory occlusion on the time course and magnitude of postexercise O2 consumption (VO2) and blood lactate responses, nine male subjects were studied twice for 50 min on a cycle ergometer. On one occasion, leg blood flow was occluded with surgical thigh cuffs placed below the buttocks and inflated to 200 mmHg. The protocol consisted of a 10-min rest, 12 min of exercise at 40% peak O2 consumption (VO2 peak), and a 28-min resting recovery while respiratory gas exchange was determined breath by breath. Occlusion (OCC) spanned min 6-8 during the 12-min work bout and elicited mean blood lactate of 5.2 +/- 0.8 mM, which was 380% greater than control (CON). During 18 min of recovery, blood lactate after OCC remained significantly above CON values. VO2 was significantly lower during exercise with OCC compared with CON but was significantly higher during the 4 min of exercise after cuff release. VO2 was higher after OCC during the first 4 min of recovery but was not significantly different thereafter. Neither total recovery VO2 (gross recovery VO2 with no base-line subtraction) nor excess postexercise VO2 (net recovery VO2 above an asymptotic base line) was significantly different for OCC and CON conditions (13.71 +/- 0.45 vs. 13.44 +/- 0.61 liters and 4.93 +/- 0.26 vs. 4.17 +/- 0.35 liters, respectively). Manipulation of exercise blood lactate levels had no significant effect on the slow ("lactacid") component of the recovery VO2.     

Gas chromatographic method using photoionization detection for the determination of breath pentane

Lipid peroxidation is thought to be an important event in the pathogenesis of atherosclerosis. It has been suggested that pentane, which can be formed during the oxidation of ω-6 fatty acids, is a marker of lipid peroxidation. Previous studies have reported elevated breath pentane and serum markers of lipid peroxidation in smokers. However, chromatographic separation of pentane from isoprene in virtually all of these studies was incomplete and the methods used did not resolve pentane into its isomers, n-pentane and isopentane. Additionally, most current methods are complicated, requiring trapping and concentrating steps to obtain adequate sensitivity prior to hydrocarbon analysis. The purpose of the current study was to develop a gas chromatographic system to analyze breath pentane, that addressses the above technical problems and that would provide a simple in vivo method for measuring lipid. n-Pentane and isopentane standards were easily separated from isoprene with a Al₂O₃/KCl capillary column contained in a portable gas chromatograph equipped with a photoionization detector. The analysis of repeated measures showed a low coefficient of variation for measurements of n-pentane (10%) and isopentane (9%). We measured breath pentane in 27 subjects (15 smokers, 12 non-smokers). There were no significant difference between the baseline and 4 week interval measurements of n-pentane for smokers both before and after cigarette smoking. The within-subject variability data showed that the assay is highly reproducible for both low and high pentane levels in smokers. Smokers were found to have higher levels of both n-pentane and isopentane than non-smokers (P<0.001). In addition, smokers had further significant elevation of pentane levels 10 min after smoking (P<0.001), which returned to baseline by 1 h. These studies demonstrate that measurement of breath pentane, using a gas chromatograph with a photoionization detector, is simple and reproducible. Additionally, these results suggest that pentane elevation associated with smoking is secondary to the oxidant effects of cigarette smoke and an important temporal relationship exists between cigarette smoking and breath sample analysis.     

The influence of dietary manipulation on plasma ammonia accumulation during incremental exercise in man

The influence of a pattern of exercise and dietary manipulation, intended to alter carbohydrate (CHO) availability, on pre-exercise acid-base status and plasma ammonia and blood lactate accumulation during incremental exercise was investigated. On three separate occasions, five healthy male subjects underwent a pre-determined incremental exercise test (IET) on an electrically braked cycle ergometer. Each IET involved subjects exercising for 5 min at 30%, 50%, 70% and 95% of their maximal oxygen uptake (VO2max) and workloads were separated by 5 min rest. The first IET took place after 3 days of normal dietary CHO intake. The second and third tests followed 3 days of low or high CHO intake, which was preceded by prolonged exercise to exhaustion in an attempt to deplete muscle and liver glycogen stores. Acid-base status and plasma ammonia and blood lactate levels were measured on arterialised venous blood samples immediately prior to and during the final 15 s of exercise at each workload and for 40 min following the completion of each IET. Three days of low CHO intake resulted in the development of a mild metabolic acidosis in all subjects. Plasma ammonia (NH3) accumulation on the low-CHO diet tended to be greater than normal at each exercise workload. Values returned towards resting levels during each recovery period. After the normal and high-CHO diets plasma NH3 levels did not markedly increase above resting values until after exercise at 95% VO2max. Plasma NH3 levels after the high-CHO diet were similar to those after the normal CHO diet.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of inspired substance concentrations on the results of breath analysis in mechanically ventilated patients

Abstract

A well-defined relationship has to exist between substance concentrations in blood and in breath if blood-borne volatile organic compounds (VOCs) are to be used as breath markers of disease or health. In this study, the impact of inspired substances on this relationship was investigated systematically. VOCs were determined in inspired and expired air and in arterial and mixed venous blood of 46 mechanically ventilated patients by means of SPME, GC/MS. Mean inspired concentrations were 25% of expired concentrations for pentane, 7.5% for acetone, 0.7% for isoprene and 0.4% for isoflurane. Only if inspired concentrations were <5% did substance disappearance rates from blood and exhalation rates correlate well. Exhaled substance concentrations depended on venous and inspired concentrations. Patients with sepsis had higher n-pentane and lower acetone concentrations in mixed venous blood than patients without sepsis (2.27 (0.37–8.70) versus 0.65 (0.33–1.48) nmol L^?1 and 69 (22–99) versus 18 (6.7–56) µmol L^?1). n-Pentane and acetone concentrations in breath showed no differences between the patient groups, regardless whether or not expired concentrations were corrected for inspired concentrations. In mechanically ventilated patients, concentration profiles of volatile substances in breath may considerably deviate from profiles in blood depending on the relative amount of inspired concentrations. A simple correction for inspired substance concentrations was not possible. Hence, substances having inspired concentrations >5% of expired concentrations should not be used as breath markers in these patients without knowledge of concentrations in blood and breath.     

Temporal association of nitric oxide levels and airflow in asthma after whole lung allergen challenge.

Exhaled nitric oxide (NO) levels are high in asthmatic subjects and increase with exacerbations. We hypothesized that higher levels of NO observed during asthma exacerbations are due to increased synthesis of NO. Exhaled NO and peak flows were measured in 11 asthmatic and 9 healthy control subjects before and after experimental asthmatic response induced by whole lung allergen challenge. Baseline peak flows of asthmatics were significantly lower than controls and decreased significantly immediately after challenge (P = 0.004). NO was measured by collecting exhaled breaths without breath hold (NO0) and after a 15-s breath hold (NO15). The rate of NO accumulation over time [parts/billion per second (ppb/s)] was calculated by DeltaNO/Deltat = (NO15 - NO0)/15, where Delta denotes a change and t is time. The NO accumulation rates in asthmatic and control subjects were similar at baseline; however, NO accumulation at 24 h increased threefold from baseline in asthmatic compared with control subjects (asthmatic subjects, 0.6 +/- 0.2 ppb/s; control subjects, 0.2 +/- 0.1 ppb/s; P = 0.01). Our study suggests that increased NO during an asthma exacerbation is due to increased synthesis, perhaps by increased expression of NO synthases.