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(Chest. 2005;128:1506-1510.)
© 2005 American College of Chest Physicians

Clinical Implications of the Ethane in Exhaled Breath in Patients With Acute Paraquat Intoxication*

Sae-Yong Hong, MD; Hyo-Wook Gil, MD; Jong-Oh Yang, MD; Eun-Young Lee, MD; Joo-Ock Na, MD; Ki-Hyun Seo, MD and Yong-Hoon Kim, MD

* From the Department of Internal Medicine (Dr. Gil), Soonchunhyang University College of Medicine, Cheonan, Korea.

Correspondence to: Hyo-Wook Gil, MD, Department of Internal Medicine, Soonchunhyang University Cheonan Hospital, 23–20 Bongmyung-Dong, Cheonan 330–100, Republic of Korea; e-mail: hwgil{at}schch.co.kr


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Pulmonary fibrosis due to lipid peroxidation is a major symptom of paraquat intoxication. Ethane in the expired breath (exEth) reflects lipid peroxidation and may be a measure of the damage effected by oxygen radicals in acute lung injury. The purpose of this study was to evaluate the clinical efficacy of exEth as a measure of exposure to paraquat and as an indicator of lung damage.

Design: Exposure levels were evaluated by the amount ingested, semiquantitative measurement of urine paraquat levels, and plasma paraquat concentration. End-tidal breath was collected for measurement of ethane 24 h after paraquat ingestion. Renal function and blood gas analyses were conducted on the same day as the breath collection, and the final clinical outcome was defined as either recovery or death. Associations between exEth and paraquat exposure profiles and clinical outcomes were assessed using linear regression models.

Patients: Twenty-one patients poisoned by paraquat were selected for the study during 2001 and 2002.

Results: exEth could not be used as a predictor of laboratory parameters such as PaO2, PaCO2, serum creatinine, and lung injury (as graded by high-resolution CT). A logistical analysis revealed that only the amount of paraquat ingested was a significant predictor of fatality (p = 0.021). The strength of the association between exEth and fatality was unaffected by the addition of potential confounders such as age, sex, and time interval and paraquat concentration.

Conclusion: exEth cannot be used as either an independent predictor of survival or a specific marker of lung injury in patients with acute paraquat poisoning.

Key Words: ethane • ethane in exhaled breath • paraquat


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Paraquat (1,1'-dimethyl-4,4'-bipyridium dichloride) was introduced in 1962 as an effective herbicide. Since then it has been discovered to have low chronic toxicity levels because of its rapid deactivation on contact with soil.1 However, even though the agricultural population is decreasing in Korea, the incidence of paraquat poisoning in humans is rapidly increasing.2 Paraquat disrupts photosynthesis in plants by inhibiting the conversion of nicotinamide adenine dinucleotide phosphate to reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by interfering with the electron-transfer system.3 A similar reduction in NADPH probably renders human cells more susceptible to attack by lipid hydroperoxidase.

The early manifestation of paraquat poisoning includes corrosive effects on the GI mucosa, liver injury, and damage to the renal tubules,4 whereas early mortality from paraquat poisoning is attributable mainly to alveolitis, which results in respiratory failure.25 In the pneumocyte, cellular injury is initiated by the NADPH-dependent reduction of paraquat to the mono-cation radical.3 Identifying the degree of free-radical-mediated damage is both a critical and complicated issue because of the multiple reactions in which free radicals may be involved and the chain reactions that may ensue. Furthermore, the favored propagation reaction varies depending on the activity of various enzymes and endogenous scavengers, the type of lipid in the cell, and the amount of oxygen available.

The severity of lung injury can be estimated by the degree of hypoxia6 and/or by pulmonary imaging techniques,7 but these methods merely reflect the end results of lung injury with no reference to the ongoing process of tissue damage mediated by the free radicals. Free radicals are evanescent. In biological settings, they are usually detected by their products, compounded with biomolecules that reflect the damage caused by the free radicals. In the interactions with lipids, free radicals initiate chain reactions of several possible products that may be used as specific evidence of lipid peroxidation. During the past decade, there has been an improvement in the methods used to analyze free-radical damage.8 The detection of hydrocarbons such as ethane or pentane in exhaled breath has attracted particular interest because these molecules are known to appear within seconds after the release of free radicals from tissues.910111213 In terms of urgency and the strength of free-radical-mediated injury, acute paraquat poisoning is a typical disease entity that leads most patients to death within a few days of acute intoxication.

In the clinical setting, IV administration of an antioxidant to paraquat-poisoned patients is mandatory to enhance their antioxidant capacity. However, from a clinical point of view, the optimum doses of antioxidants such as N-acetyl cysteine, glutathione, and vitamin C have yet to be defined. One of the reasons for this is the persisting difficulty in identifying the degree of ongoing free-radical-mediated tissue injury.

If the analysis of ethane in the expired breath (exEth) of paraquat-poisoned patients helps define the size and the timing of free-radical appearance,14 it could also help us to evaluate the efficacy of intervention protocols that ameliorate free-radical-mediated injury. The aims of this study are to evaluate the correlation between exEth and patient outcome, and to verify whether it would be a possible independent predictor of survival in patients with acute paraquat poisoning.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This study was designed to compare the exEth obtained 24 h after paraquat ingestion and to determine the relationship between that exEth and the clinical outcome, including lung injury as assessed by high-resolution CT (HRCT) and exposure variables.

Patients
Twenty-one patients poisoned by paraquat were enrolled in this study. All of the subjects were ingestion cases with clear mentality who were admitted to the Institute of Pesticide Poisoning, Soonchunhyang University Hospital, Cheonon, Korea, from January 2001 through to December 2002. The degree of exposure to paraquat was assessed as soon as the patient arrived at the emergency department (ED) by the amount ingested (where one mouthful = 20 mL), the urine paraquat test (dithionite method), and plasma paraquat concentration (as assessed by high-performance liquid chromatography), as described in detail elsewhere.15 Standardized medical emergency procedures were conducted as follows. Briefly, after gastric lavage, 100 g of Fuller earth in 200 mL of 20% mannitol was administered if intoxication had occurred within 3 h of hospital admission. Emergency hemoperfusion (adsorba 300; Gambro Dialysatoren; Hechingen, Germany) was performed until the urinary paraquat test result was negative.15 All of the samples taken for this study were obtained with the permission and informed consent of either the subjects or their guardians.

Clinical outcome was defined as survivor or nonsurvivor. In our experience,6 the survivors can be described as patients who survived for > 4 weeks following paraquat ingestion and whose arterial blood gas analysis, serum creatinine, and liver function test findings were all within the normal range at discharge from the hospital.6 The survivors were discharged after 4 weeks in the hospital in a healthy state and without any abnormalities in CBC count, urinalysis, blood chemistry, arterial blood gas analysis, ECG, and chest radiography.

Measurements
HRCT was performed 7 days after paraquat ingestion, as recommended by the results of a preliminary study.7 Grading of paraquat-affected lungs using HRCT was classified according to the area of lung that was injured, as follows: +, < 25%; ++, 25 to 50%; +++, 50 to 75%; and ++++, 75 to 100% of the total lung area.7

Breath Collection
End-tidal breath samples were collected 24 h after paraquat ingestion. Since most of the patients with acute paraquat intoxication faced the emergency situation of an imminent extinction of life, and hence ethical considerations allowed only emergency therapy to be performed on them during the first 24 h, their exhaled breath samples were collected only when their vital signs had stabilized and they were able to cooperate voluntarily. The patients were placed in a supine position and asked to inhale and exhale through a disposable mouthpiece and respiratory membrane filter. After a short practice, end-tidal exhaled breath was collected from the expiratory arm of this valve, through a two-way nonrebreathing valve, to a 1-L bag (Tedlar; SKC; Eighty Four, PA).

Measurement of exEth
The samples of end-tidal air from the patients and 10 healthy control subjects (nonsmoking men aged 25 to 30 years) were purged with ethane-free nitrogen. The stability of ethane in Tedlar bags was confirmed by the absence of a measurable change in the concentration of the gas in them over a 2-week period.

The exEth was analyzed using a homemade cryogenic concentration gas chromatography (GC)-flame ionization detector analysis system, which was built by staff at the Korea Research Institute of Standards and Science, Daejeon, Korea. A six-port gas-switching valve (SV) was modified for use with this system. Instead of a gas-sampling loop, a glass-bead-filled U-shaped tube was used for the concentration trap. Liquid oxygen was used as a cooling medium, and hot water at 75°C was used for heating the tube during the desorption step. A breath-air sample was pumped through the concentration loop at a rate of 50 mL/min, and a 50-mL air sample was concentrated. The SV, transfer lines between the bag sample and the SV, and transfer lines between the SV and GC column were heated to 105°C to avoid moisture condensation. After desorption of the U tube, it was baked at 120°C.

A GC column (GC-Q; J&W Scientific; Folsom, CA) with a length of 30 m and an internal diameter of 0.542 mm was used for ethane separation. The carrier gas flow was maintained at a rate of 4 mL/min. The GC oven temperature was changed from 60°C (5 min) to 230°C (5 min) at a rate of 15°C/min. Ethane was eluted at 3.17 min and detected by a flame ionization detector that was set at a temperature of 260°C. This analysis system provides a quantitative detection limit for ethane of 0.1 parts per billion (ppb) [obtained by multiplying by 10 the repeatability of 1 ppb ethane standard gas], and a 1% repeatability was obtained for triplicate measurements of the standard gas. Standard gas prepared gravimetrically by the Korea Research Institute of Standards and Science was used as the mother standard gas at 1,268 ppm. The gas was diluted to ppb concentrations and used as the calibration gas for the sample analysis. The linearity of the analysis condition also was checked at concentration levels of 1 to 10 ppb, and showed very good linearity (0.9999).

Statistical Analysis
Data are expressed as the mean (SD) or as a percentage of the control value, and differences were compared using statistical models. Relationships between the levels of exEth and exposure variables, clinical outcomes, and lung injury (as estimated by HRCT) were assessed. Regression analyses were applied to adjust for potential confounders, and associations are presented as odds ratios or ß-coefficients.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Even though the level of exEth was higher in the nonsurvivor group than in the survivor group, it is neither an independent predictor of survival nor a specific marker of lung injury. The subjects (71.4% female; mean age, 41.3 years; SD, 13.7) were admitted to the ED on average 12.5 h (SD, 13.1) after a mean paraquat ingestion of 46.7 mL. All subjects presented a positive urine paraquat test result. Plasma paraquat concentrations ranged from 0.10 to 5.10 µg/dL, and the mean level of exEth was 5.89 ppb (SD, 1.77; range, 1.1 to 10.0 ppb). A comparison of the parameters of the survivor and nonsurvivor groups is summarized in Table 1 . The exEth was 3.4 ppb (SD, 0.8) in the healthy control subjects. The exEth was higher in the nonsurvivor group than in the survivor group: 7.30 ppb (SD, 1.96) vs 5.07 ppb (SD, 0.98), p = 0.012 (Fig 1 ).


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  		Table 1.. Comparison of Paraquat Poisoning Characteristics Between the Survivor and Nonsurvivor Groups*

 


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  		Figure 1.. Figure 1. Comparison of exEth levels (p = 0.012).

 
Plasma paraquat concentration was significantly correlated with urine paraquat concentration (r = 0.537, p = 0.012) but not with the exEth (r = 0.236, p = 0.302). The amount of paraquat ingested and the concentration of paraquat in both the plasma and urine were not related to the levels of exEth. In addition, exEth levels were not predictors of laboratory parameters such as PaCO2, PaO2, serum creatinine, and lung injury (as graded by HRCT).

The logistic analysis revealed that only the amount of paraquat ingested was a significant predictor of fatality (p = 0.021; Table 2 ). The strength of the association between exEth and fatality was unaffected by the addition of potential confounders such as age, sex, time interval between ingestion of paraquat and treatment, and plasma paraquat concentration (data not shown).


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  		Table 2.. Association Between Fatality and Each Exposure Variable*

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results indicate that even though the level of exEth was higher in the nonsurvivor group than in the survivor group, it is neither an independent predictor of survival nor a specific marker of lung injury in patients with acute paraquat poisoning. The major cause of death from paraquat poisoning is respiratory failure due to oxidative insult to the alveolar epithelium.16 It would therefore be very useful to identify markers that indicate the progress of lung injury in paraquat intoxication. Of course, there are already powerful tools available with which to estimate the degree of lung damage in these cases, such as HRCT of the lung, arterial blood gas analysis, and pulmonary function tests. However, the results of these studies do not reflect the ongoing process, but merely the end result of the progression to lung injury mediated by paraquat.

Kazui et al17 showed that the exEth reflects in vivo lipid peroxidation. Therefore, it was possible that the analysis of exEth would provide a way of quantifying the magnitude and timing of the appearance of free radicals.14 In our preliminary study, levels of ethane in a Tedlar bag were very stable, showing no detectable change in concentration over a 2-week period. Breath-air samples containing large amounts of moisture can be analyzed with a high level of repeatability (1 to 6%; average, 3%). Good linearity was also obtained at concentrations in the low-ppb range. In our preliminary study, it was noted that the level of exEth is very sensitive to the environment, especially to whether or not the individual smoked (data are not presented). However, in the study presented herein, the focus was merely to observe the relationship between the exEth and clinical parameters in patients with acute paraquat intoxication, and all exhaled breath samples were derived from the same ICUs.

To our knowledge, the clinical trial reported herein represents the first attempt to evaluate the exEth as a clinical marker of the degree of lung damage following acute paraquat poisoning. Our finding of a correlation between exEth and survival rate may be helpful in understanding the progression of reactive oxygen species-induced lung injury in patients with acute paraquat intoxication. Our study confirmed the previous finding18 that the outcome after acute paraquat poisoning is determined by the degree of paraquat exposure and plasma paraquat concentration. As compared to the Proudfoot survival curve of plasma paraquat, the agreement rate was 66.6% (14 of 21), with a 0.276 {kappa} index.

Our study does have several limitations, although we believe that these do not invalidate its conclusions. First of all, there is no evidence that all of the exEth is from the lung tissue. Theoretically, paraquat-induced lung injury could involve multiple organs. Therefore, it is reasonable to assume that most of the organs and/or tissue would produce ethane, and that this ethane could be evaporated via the lung. However, it is well known that paraquat has a higher affinity for alveolar cells than for any other tissue or cell type. Taken together with the fact that the lung is the most vulnerable organ during paraquat intoxication, we believe that a significant proportion of the exEth originates from the lung tissue itself.

Contrary to our expectations, there was no significant relationship between the exEth and plasma paraquat concentration. One factor we have to consider before drawing a conclusion from this is that the time interval between paraquat ingestion and measurement of plasma paraquat levels at the ED was not uniform between cases. Theoretically, the sooner the sampling time for the exEth is the better because the paraquat-induced lung injury progresses rapidly. Further, continuous timed measurements from earliest possible would provide more informative results. However, as we mentioned earlier, most patients with acute paraquat intoxication faced the emergency situation of imminent death. Therefore, from the ethical point of view, the emergency therapy such as hemoperfusion, forced diuresis, and IV administration of antioxidants should be performed before any kind of research work during the first several hours. That was the reason why we set the sampling time from the 24 h after paraquat ingestion. A controlled study utilizing an experimental animal model is necessary to clarify this result. The determination of exEth is subject to error due to reactions that may occur during sampling and because some patients suffered from shortness of breath, and even with considerable effort it was difficult for them to produce an appropriate expiratory reserve volume.

No significant relationship was observed between the severity of lung injury and exEth, even though the latter was higher in the nonsurvivor group than in the survivor group. This implies that the exEth measured 24 h after paraquat ingestion is not a marker of lung injury in paraquat intoxication. Another possibility is that this discrepancy was caused by the way we performed the lung HRCT. In our previous clinical experience, the sensitivity and/or specificity of this test was valid only when lung HRCT was performed a minimum of 7 days after paraquat ingestion.7 Unfortunately, 14 patients in the present study had already died by this time. This suggests that lung injury occurs early in the poisoning process and is complete within the first few days after paraquat ingestion. In conclusion, even though the exEth levels were higher in the nonsurvivor group than in the survivor group, they cannot be used as either an independent predictor of survival or a specific marker of lung injury when it is measured 24 h after acute paraquat poisoning.


   Footnotes
 
Abbreviations: ED = emergency department; exEth = ethane in the expired breath; GC = gas chromatography; HRCT = high-resolution CT; NADPH = reduced nicotinamide adenine dinucleotide phosphate; ppb = parts per billion; SV = six-port gas-switching valve

This study was supported by a grant from Syngenta Korea.

Received for publication September 7, 2004. Accepted for publication February 23, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  18. Hart, TB, Nevitt, A, Whitehead, A A new statistical approach to the prognostic significance of plasma paraquat concentrations. Lancet 1984;2,1222-1223[ISI][Medline]




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