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European Journal of Heart Failure 2001 3(4):423-427; doi:10.1016/S1388-9842(01)00128-3
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© 2001 European Society of Cardiology

Breath isoprene in patients with heart failure

Lawrence T. McGratha,*, Robin Patrickb and Bernard Silkea

a Department of Therapeutics and Pharmacology, The Queen's University of Belfast 97 Lisburn Road, Belfast BT9 7BL, Belfast, N Ireland, UK
b Department of Chemistry, The Queen's University of Belfast 97 Lisburn Road, Belfast BT9 7BL, Belfast, N Ireland, UK

* Corresponding author. Tel.: +44-2890335770; fax: +44-2890438346. E-mail address: l.mcgrath{at}qub.ac.uk (L.T. McGrath).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Statistical analyses and...
 4. Results
 5. Discussion
 References
 
Background: Chronic heart failure (CHF) is characterised by increased vascular resistance. This increased after load on the left ventricle contributes to the vicious cycle that leads to progression of myocardial failure, multiple organ failure and death. There is evidence for increased oxidative stress in heart failure, which will influence the myocardium but also peripheral vasculature endothelium.

Aims: The aim of the present study was to examine the production of isoprene, reputed to reflect oxidative stress, in patients with CHF compared to control subjects.

Methods: Twelve patients with CHF and thirty-one healthy control subjects free from heart disease were studied. Breath was collected via a two-way non-re-breathing valve into a 60-l gas collection bag. A sample of ambient air was collected at the same time. A measured aliquot of patient breath and ambient air (approx. 1.5 l) was adsorbed onto a gas adsorption tube packed with poropak-Q. Isoprene was measured using GC/MS and the production rate calculated. All samples of breath were collected at 10.00 h after subjects had been sitting at rest for 15 min.

Results: Breath isoprene production in subjects with CHF was significantly reduced compared to controls 83(23) vs. 168(20) pmol min–1 kg–1.

Conclusion: Breath isoprene does not directly reflect oxidative stress in CHF.

Key Words: Heart failure • Isoprene • Breath • Oxidative stress

Received September 21, 2000; Revised November 23, 2000; Accepted January 17, 2001


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Statistical analyses and...
 4. Results
 5. Discussion
 References
 
The underlying abnormality in heart failure is a failure in the myocardium itself. Impaired ability of the left ventricle to pump blood is aggravated by impaired ability of the peripheral vasculature endothelium to dilate resulting in increased load upon the failing left ventricle. Persistent left ventricular dysfunction and the resulting ischaemia in vital organs due to decreased perfusion sets off a series of reflex responses in an attempt to maintain perfusion [1]. These mechanisms include neurogenic and humoral pathways with increased sympathetic activity and increased release of catecholamines, activation of the renin–angiotensin system, vasopressin, endothelin-1 and neuropeptide Y [2]. These responses lead to vasoconstriction, fluid and electrolyte accumulation and LV remodelling. This sequence of events is responsible for the ‘vicious cycle’ [1] that occurs in heart failure and leads to progression of myocardial failure, multiple organ failure and death.

A consistent theme in heart failure is impaired perfusion of the myocardium, and other major organs, with frequent cycles of ischaemia/reperfusion. Reperfusion of ischaemic tissue has been shown to cause damage to the tissue by oxidative stress [3]. Oxidative stress has been proposed to play a role in the genesis and progression of acute and chronic heart failure [4,5]. Numerous technologies exist for the assessment of oxidative stress. Reduced plasma levels of endogenous antioxidants and increased levels of plasma lipid peroxides (produced by free radical damage to polyunsaturated fatty acids) have been found in heart failure [6]. These lipid hydroperoxides fragment to produce numerous degradation products. One of the products is believed to be the volatile isoprene (2-methyl-1,3 butadiene), which can be detected in breath [7]. In this study we examined the level of breath isoprene in patients with heart failure.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Statistical analyses and...
 4. Results
 5. Discussion
 References
 
2.1. Subjects
Twelve patients, 11 male aged 60 (55–65) years and 1 female aged 62 years were recruited from patients attending the heart failure clinic of the Belfast City Hospital. Patients compensated chronic heart failure and were in the New York Heart Association classification symptom grades II and III. Thirty-one healthy control subjects free from heart disease, 21 males aged 40 (35–45) years and 10 females aged 38 (30–46) years were recruited from the local community. The study was approved by the local ethics committee and conformed to the principles outlined in the Declaration of Helsinki.

2.2. Sample collection
Breath was collected via a two-way non-re-breathing valve into a 60-l gas collection bag [8]. The subjects nose was clamped and the collection tubes equilibrated for 60 s, the breath being vented to air via a three-way tap. The breath was then transferred into the collection bag and collected for 3 min and the volume recorded. A sample of ambient air was collected at the same time. A measured aliquot of patient breath and ambient air (approx. 1.5 l) was drawn through a gas adsorption tube packed with poropak-Q. All samples of breath were collected at 10.00 h after subjects had been sitting at rest for 15 min.

2.3. Breath analysis
The adsorbed organic breath contents were analysed for isoprene by thermal desorption gas chromatography/mass spectrometry using the following procedure.

The analytical system comprised a Perkin-Elmer ATD 400 automated thermal desorption unit, coupled to a Hewlett Packard 5890 gas chromatograph with a Restek RTx% 30-m fused silica capillary column. The column was directly interfaced to a VG Trio bench-top mass spectrometer. The sample tubes were purged at 200°C for 10 min and the desorbed components collected on a cold trap packed with Tenax and Maintained at –30°C. The cold trap was ballistically heated to 250°C, which initiated the GC. The column was held at 40°C for 5 min and then heated at 10°C/min to a final temperature of 280°C. Isoprene, which eluted after approximately 3 min, was detected by monitoring the ion at m/z 68. Levels were quantified relative to toluene, which was introduced on to a tube by direct injection in methanol. The concentration of isoprene in breath was used to calculate the rate of production per minute. This was then related to body mass and expressed as pmol min–1 kg–1.


   3. Statistical analyses and expression of results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analyses and...
4. Results
 5. Discussion
 References
 
Data were analysed using the SPSS package (SPSS Inc, Chicago, IL, USA). Comparisons between the two groups (patients during exacerbation and controls) were made using the Mann–Whitney rank sum test. Absolute values are summarised as means (S.E.). A P-value of 0.05 or less was considered significant.


   4. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analyses and...
 4. Results
5. Discussion
 References
 
Breath isoprene concentrations are summarised in Fig. 1. Isoprene was calculated as a concentration, nmol l–1 and as a weight adjusted production rate, pmol min–1 kg–1. The concentration of isoprene in the breath of heart failure patients was significantly lower than that found in controls [0.66 (0.20) vs. 1.12 (0.14) nmol l–1, P<0.05]. The rate of production of isoprene was also significantly lower in patients compared to controls [83.3 (23.2) vs. 168 (19.8) pmol min–1 kg–1, P<0.05].


Figure 1
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  		Fig. 1 Breath isoprene production rate (a) and concentration (b) in healthy controls and individuals with congestive heart failure. Breath was collected from resting, fasting subjects over a period of 3 min. Allowance was made for ambient levels of isoprene (not detected) for each subject. All values are means±S.E.M. Concentration and production rate in CHF patients were significantly lower than in controls P<0.05.

 

   5. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analyses and...
 4. Results
 5. Discussion
References
 
Isoprene is one of the most abundant hydrocarbons in human breath [9]. Analysis of breath isoprene, however, has inherent difficulties. Measurement of volatile hydrocarbons in breath has been used in a number of conditions [10–12]. The original methods had unacceptable limitations of sensitivity and specificity with isoprene being mistakenly identified as pentane [13]. Kohlmuller and Kochen developed a gas chromatographic/mass spectroscopic (GC/MS) method eliminating these problems [13]. Analysis of isoprene has inherent difficulties apart from methodology. These include a wide distribution within and between individuals, a distinct circadian rhythm and dependence on state of nutrition and exercise [14,15]. Isoprene increases with age and is independent of metabolic state in diabetic children [16].

In an effort to minimise variations all of the subjects in our study were closely matched, sampled fasting at the same time of day, and were resting. A further problem arises with the units used. Some workers measure concentration in expired air (mass/volume). This is confounded by rate and depth of breathing and non-alveolar air which has not undergone gas exchange. To minimise variations isoprene was calculated as a production rate in mass eliminated per kg of body mass per unit of time rather than as an absolute concentration.

The evidence for increased oxidative stress in heart failure is convincing [17–19]. Doubts about the technology used in these studies have been partly removed by the use of more specific assays [20]. While isoprene has been reputed to reflect oxidative stress its source is not completely understood. Phillips et al. suggested that breath isoprene was from an external source, that is an air pollutant [21]. We found no evidence of isoprene in contemporaneously collected ambient air. Taucher et al. have shown that isoprene is not derived non-enzymatically from allylic C5 hydrocarbons [9]. Foster et al. [22] demonstrated increased breath isoprene in the breath of young adults exposed to ozone, a treatment expected to cause oxidative damage to the lung surface. There is evidence that breath isoprene may be related to sterol synthesis. Kohlmuller and Kochen [13] argue that polyisoprenes, e.g. squalene, are possible sources of isoprene via radically mediated in vivo peroxidation. This was supported by Stein and Mead who produced evidence, in vitro, that peroxidation of squalene could produce isoprene at low O2 partial pressures [23]. There is compelling evidence that breath isoprene is derived from a mechanism not involving oxidative stress however. Euler et al. showed increased breath pentane but unchanged isoprene following smoking [24]. Mendis et al. demonstrated an increase in breath isoprene, but not pentane, in patients experiencing acute myocardial infarction suggesting the increased isoprene was from some source other than increased oxidative stress [25]. The work of Schubert et al. is interesting in this respect [26]. While they demonstrated a decrease in breath isoprene in critically ill patients who developed pulmonary infection, breath pentane actually increased. These conflicts can only be resolved by measuring isoprene in blood.

The crucial issue raised in the present study was that breath isoprene was decreased in a condition associated with increased oxidative stress. Stone et al. have suggested that isoprene might also be produced from mevalonate as a by-product of cholesterol synthesis [27]. They provided evidence to support this hypothesis by manipulating cholesterol synthesis in human volunteers and comparing breath isoprene with rate of cholesterol synthesis. The parallel decreases in isoprene excretion and cholesterol synthesis caused by these pharmacologic means suggest that breath isoprene is derived from the cholesterol synthesis pathway. Deneris et al. were the first to provide evidence that isoprene was linked to cholesterol synthesis showing that isoprene can be produced from mevalonate in the cytosolic fraction of isolated rat liver [28].

Memon et al. administered endotoxin, tumour necrosis factor, and interleukin-1 to animals and showed a marked decrease in mRNA levels of squalene synthase with a decrease in squalene synthase activity and protein mass [29]. These actions are mediated by TNF-{alpha} [30]. The resultant decrease in squalene limits substrate availability with resultant decrease in isoprene production. During the host response to infection and/or inflammation regulation of squalene synthase by cytokines may have effects on the regulation of substrate flux into the non-sterol pathway of mevalonate metabolism [30]. In this respect we have demonstrated decreased breath isoprene in cystic fibrosis patients during acute respiratory exacerbation [31]. While individuals demonstrated an increased degree of oxidative stress compared to periods of non-respiratory exacerbation and controls they also had elevated levels of cytokines. This relationship between isoprene, squalene synthesis and the inflammatory process has been strengthened by recent work [15,32]. TNF-{alpha} is released by the immuno-response to heart failure. In addition to its impact on sterol synthesis, it causes increased oxidative stress by three possible mechanisms: inducing nitric oxide synthase with increased production of nitric oxide, itself a free radical; increased production of superoxide anions in the mitochondria; and reduction of anti-oxidant defences [33]. The finding of low breath isoprene in heart failure may thus be a combination of reduced sterol synthesis and peroxidation of a reduced pool of squalene by free radicals.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Statistical analyses and...
 4. Results
 5. Discussion
 References
 

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