INTRODUCTION

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Keywords: air pollution; particulate matter; lung diseases; bronchoalveolar lavage fluid

Epidemiological investigation has established an association between exposure to particulate matter (PM) with both human mortality and diverse indices of human morbidity. The United Nations and World Health Organization estimate the number of premature deaths resulting from PM exposure to be 500,000 per year worldwide. Similarly, respiratory symptoms, school absences, bronchodilator use among patients with asthma, and hospitalization rates for bronchitis and pneumonia all increase with PM exposure while values of peak expiratory flow rate decrease. In addition to a respiratory injury, evidence has been reported to support both changes in blood indices and cardiac effects after particle exposure. Changes in blood levels of C-reactive protein (1), fibrinogen (2, 3), factor VII (1), and red blood cells (1) are found after inhalation of elevated concentrations of PM. Pulse rate (4, 5), arrhythmia induction in patients with implanted cardiac defibrillators (6), and heart rate variability (7) can similarly correspond to concentrations of ambient air particles.

The designation of those individuals with mortality and morbidity specifically resulting from PM exposure is implausible as a result of the lack of an accurate marker of PM exposure. Subsequently, their clinical presentation remains unclear. We describe a patient with respiratory damage, abnormal blood end points, and a cardiac effect following exposure to an emission source air pollution particle. In addition, we chemically characterize the particle, quantify the oxidants generated in vitro by the PM, and describe the biological effect in an animal model.

	CASE REPORT

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A 42-yr-old, unemployed male (lifetime nonsmoker) with an 8-yr history of diabetes mellitus attempted to clean his home oil-burning furnace, located in his living room, using a conventional vacuum cleaner. After removing almost all oil fly ash from the furnace, he noted suspended particles in the room and discontinued vacuuming. The patient remained indoors following the vacuuming of the particles. Twenty-four hours later, shortness of breath, a nonproductive cough, and wheezing developed. These complaints worsened over time. One week after exposure, the patient presented to an urgent care facility and was treated with nebulized albuterol. There was little improvement in his condition and chills developed. Two weeks after exposure, the patient presented to the Emergency Department at the University of North Carolina with shortness of breath and fevers. The physical examination was remarkable for obesity (weight of 339 pounds; height of 66 inches), a temperature of 38.4° C, diminished breath sounds at both bases, and pitting edema. Laboratory values included a white blood cell count of 5400/mm³ and an erythrocyte sedimentation rate (ESR) of 47 mm/h. Titers for rheumatoid factor, antinuclear antibody, and antineutrophilic cytoplasmic antibody were negative and complement levels were normal. The oxygen saturation was 91% on room air. The chest X-ray demonstrated a left lower lobe infiltrate (Figure 1A). The patient was unable to do pulmonary function tests but peak flow measurements varied between 140 and 200 L/min after bronchodilator. Blood cultures were negative. The patient was given a presumptive diagnosis of community acquired pneumonia and therapy was initiated with cefotaxime, azithromycin, oxygen, and nebulized albuterol.

View larger version (106K): [in this window] [in a new window]   Figure 1.   A posteroanterior chest X-ray demonstrated a left lower lobe infiltrate (A). Three days later, both the chest X-ray and CT revealed diffuse opacities (ground glass) throughout both lung fields (B). Two weeks after discharge, the chest X-ray was normal.

The patient's condition rapidly deteriorated with worsening shortness of breath and an increasing oxygen requirement (100% by facemask) hours after admission. Myocardial infarction was excluded from the differential employing blood enzymes. The chest X ray showed bilateral ill-defined infiltrates while the computed tomograph showed ground glass opacities bilaterally (Figure 1B). Antibiotic coverage was switched to vancomycin, piperacillin/tazobactam, and ciprofloxacin. Legionella pneumophilia serogroup antigen in the urine was negative. On the third day after admission, bronchoscopy revealed no endobronchial lesions while lavage showed a neutrophilic inflammatory response (50%). All cultures of the lavage were negative and cytopathology identified no malignant cells, pneumocystis, or fungi. A thoracoscopic wedge biopsy of the left upper lobe (lingula) and left lower lobe was performed on the fifth day. Stains and cultures for bacteria, fungi, and mycobacteria were negative. Histopathology demonstrated a dense infiltrate of macrophages without a significant number of neutrophils (Figure 2A). Numerous macrophages contained sequestered particles (Figure 2B). In addition, there was a marked hyperplasia of type II pneumocytes with hyaline membrane formation (Figure 2C). Areas also showed pulmonary edema (Figure 2D). A pathological diagnosis of an exudative phase diffuse alveolar damage with focal organization was made. The features were felt to be consistent with an injury to the lower respiratory tract occurring between 1 and 4 wk prior to biopsy.

View larger version (131K): [in this window] [in a new window]   Figure 2.   Pathological examination of biopsied lung revealed a monocytic inflammatory injury with intraalveolar macrophages but few neutrophils (A [top left]). Macrophages included pigmented PM (B [top right]). Areas of tissue exhibited features of an exudative phase of diffuse alveolar damage with organization (C [bottom left] ). Pulmonary edema was also a prominent feature in areas of the section (D [bottom right]).

The patient required intubation after the biopsy procedure for hypoxemic respiratory failure. All antibiotics were stopped immediately after biopsy and he was placed on prednisone 60 mg by mouth each day. Clinical and radiographic improvement was apparent within 36 h. After 5 d of ventilatory support, he was extubated and the chest tube was removed. Five days later, the patient was discharged on prednisone 60 mg each day to be tapered.

Shortness of breath with minimal exertion and a nonproductive cough persisted after returning home. The ESR (58 mm/h) and C-reactive protein (2.5 mg/dl; normal 0 to 2.0 mg/dl) were both elevated 4 mo after discharge. Six months after discharge, the ESR remained elevated (40 mm/h), fibrinogen was increased (546 mg/dl; normal 150 to 504 mg/dl) while viscosity was normal. Prothrombin time was elevated (1 d only) while platelets were significantly decreased during the admission. Pulmonary function tests demonstrated a restrictive lung process that persisted (Table 1).

One week after discharge, the patient noted the onset of a new, substernal chest pain with exertion. This was described as a "pressure-like" sensation without radiation that occurred twice a day, lasted minutes, and was relieved by sublingual nitroglycerin. There was no nausea, vomiting, or diaphoresis. The patient had numerous premature ventricular contractions noted on an electrocardiogram. A treadmill test had to be terminated early because of dyspnea. An adenosine thallium study demonstrated areas of moderate hypokinesis at the apex and an ejection fraction of 45%. Isosorbide dinitrate, furosemide, amlodipine besylate, and aspirin were initiated. As a result of a past medical history of diabetes and hypertension and a family history of heart disease at an early age, coronary angiography was done that disclosed no significant narrowing of the epicardial coronary arteries and an ejection fraction of 51%. Right heart pressures could not be determined. The perfusion scan was interpreted as completely normal excluding the possibility of pulmonary embolism. An echocardiogram confirmed left ventricular dysfunction. Ejection fractions of the left and right ventricles were found to be 45% and 51%, respectively, by a radionuclide ventriculogram. Further history was obtained that revealed symptoms suggestive of sleep-disordered breathing. A formal sleep study provided a diagnosis of severe obstructive sleep apnea with significant desaturations during apneic episodes.

	METHODS

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Ash was collected from the patient's home furnace. Carbon, hydrogen, nitrogen, and mineral ash content of the oil fly ash were analyzed by elemental analysis (Galbraith Labs, Knoxville, TN). Metal analysis was performed using inductively coupled plasma emission spectroscopy (ICPES; Perkin Elmer 40, Norwalk, CT) after acid wash in 1 N HCl. Particulate material from the furnace was mounted on a graphite specimen mount using adhesive backing. The material was viewed and images acquired in a Cambridge S200 scanning electron microscope operating at 20 kV. X ray analyses were performed using a Kevex 7000 system interfaced to the microscope with a 100 s acquire time. In vitro oxidant generation by the oil fly ash was measured employing thiobarbituric acid (TBA) reactive products of deoxyribose.

The treatment and care of animals were conducted under the direction of the Institute for Animal Care and Use Committee. Sixty-day-old, male Sprague-Dawley rats were exposed to either saline or oil fly ash (n = 10/concentration of exposure). After anesthesia with halothane (Aldrich Chemicals, Milwaukee, WI), either 0.5 ml of normal saline or a concentration of oil fly ash in 0.5 ml saline was tracheally instilled. Twenty-four hours after the exposure, rats were again anesthetized and blood was collected. Animals were euthanized and tracheally lavaged. Lavage concentrations of macrophage inflammatory protein 2 (MIP-2) were assayed using a commercially available ELISA (R&D, Minneapolis, MN). Neutrophils were expressed as the percentage of total cells recovered. Lavage protein was determined using the Pierce Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL).

Data are expressed as mean values ± standard error. Differences between multiple groups were compared using one-way analysis of variance. The post-hoc test employed was Scheffe's test. Significance was assumed at p < 0.05.

	RESULTS

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Chemical characterization of the particle revealed the following (mean ± standard deviation): carbon 25.3 ± 8.6%, hydrogen 0.9 ± 0.3 %, nitrogen <0.05%, oxygen 14.4 ± 3.0%, and sulfur 2.2 ± 0.5%. Ionizable metal concentrations and sulfate content associated with the ash (µg/g particle) are iron 24,892 ± 84, lead 441 ± 19, copper 164 ± 4, zinc 31.6 ± 0.3, nickel 20.4 ± 6.8, vanadium 6.0 ± 2.9, chromium 3.6 ± 3.1, arsenic 2.7 ± 9.8, and sulfate 44,079 ± 242. Electron microscopy revealed particles with a range of diameters within values considered respirable (Figure 3). X ray analyses confirmed detectable levels of silica, sulfate, and iron. The ash did support in vitro electron transport and subsequently generated oxidants measured as TBA reactive products of deoxyribose (Figure 4). This oxidant generation was decreased by both the metal chelator deferoxamine (Ciba Pharmaceutical, Summit, NJ) and the radical scavenger dimethylthiourea (DMTU).

View larger version (164K): [in this window] [in a new window]   Figure 3.   Aggregates and individual particles on electron microscopy.

View larger version (19K): [in this window] [in a new window]   Figure 4.   In vitro TBA production by the oil fly ash. The reaction mixture containing 1.0 mM deoxyribose, 1.0 mM H2O2, 1.0 mM ascorbate, and oil fly ash was incubated in saline at 37° C for 60 min with agitation and then centrifuged at 1200 × g for 10 min. One milliliter of both 1.0% (wt/vol) TBA and 2.8% (wt/vol) trichloroacetic acid was added to 1.0 ml of supernatant, heated at 100° C for 10 min, cooled in ice, and the chromophore concentration determined by its absorbance at 532 nm. There was a significant increase in oxidant generation with exposure to the ash. This was inhibited by inclusion of both deferoxamine (DEF) and dimethylthiourea (DMTU) in the reaction mixture.

Intratracheal instillation of the particle in rats was associated with increased plasma concentrations of fibrinogen (141 ± 3.5 mg/dl and 168 ± 4.4 mg/dl in saline- and 500 µg ash-exposed animals, respectively, 24 h following instillation; p < 0.05) but not C-reactive protein (35.5 ± 3.9 mg/L and 35.7 ± 4.1 mg/L in saline- and 500 µg ash-exposed animals, respectively, 24 h following instillation). The exposure to the ash caused an increase in lavage concentrations of MIP-2 (Figure 5A). A neutrophilic influx into the lower respiratory tract was evident (Figure 5B). Finally, an injury was observed with elevations in lavage protein (Figure 5B).

View larger version (15K): [in this window] [in a new window]   View larger version (28K): [in this window] [in a new window]   Figure 5.   The animal model verified an inflammatory injury after exposure to the oil fly ash. There were increases in the proinflammatory mediator MIP-2 24 h after instillation of the particle (A) and a neutrophilic influx was also evident at this same time (B). Comparable to the influx of inflammatory cells, an injury followed exposure of the rats to the particle, which was reflected by increases in concentrations of lavage protein (B). *p < .05 in comparisons relative to saline instillations.

	DISCUSSION

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The particle this patient was exposed to included metals that catalyzed oxidant generation in vitro. It had a diameter allowing it entry to the lower respiratory tract. There were several factors that could potentially increase susceptibility of this patient to lung injury after particle exposure including diabetes, morbid obesity, and obstructive sleep apnea. The inhalation of the particle was associated with symptoms of respiratory compromise and pathology consistent with diffuse alveolar damage. Exposure of an animal model resulted in similar proinflammatory events and biological effect. These events are comparable to those described in controlled exposures of human subjects to PM with a release of proinflammatory mediators occurring early (10) followed both an influx of neutrophils (2, 3) and a tissue injury (2). However, the magnitude of the inflammatory injury is far greater in this specific individual, likely reflecting the intensity of the exposure.

The patient may have also had cardiac effects of PM exposure. These included a recurring "angina-like" chest pain with normal epicardial coronary arteries proven by angiography. Ambient air pollution particles have been demonstrated to transiently increase pulse rate (1, 5) and decrease heart rate variability (4). In addition, an induction of arrhythmias in patients with implanted defibrillators has been reported (6). The mechanism by which PM affects such change is not known but could include direct transport of the PM (or specific components) to the heart, release of inflammatory mediators that could interact with cardiac tissue, and neurogenic reflexes. On the basis of either an immediate release of mediators or a neurogenic reflex, it is difficult to explain why the patient continues to have cardiac complications several months after exposure unless extensive damage resulted. Distribution of inorganic fibers and particles to extrapulmonary sites support a possible transport of PM or components directly to the heart. Similarly, epidemiological investigation has noted alterations in blood components after PM exposure. Red blood cells (7), white blood cells (7), serum viscosity (7, 11), C-reactive protein (7), fibrinogen (8, 9), and factor VII (7) have been noted to change with PM. The ESR, C-reactive protein, and fibrinogen were altered in this patient exposed to oil fly ash. In addition, prothrombin time also increased and platelets decreased suggesting a coagulation defect. Changes in blood indices can reflect an acute phase response to the lung injury. The particle or its components could be transported to the liver to effect these changes. Alternatively, there are cells in the respiratory tract that could express inflammatory mediator to affect these proteins or they could be produced by cells resident in the respiratory tract itself.

In conclusion, we report a patient who presents with pulmonary damage, changes in blood indices, and a possible cardiac injury after an exposure to an emission source PM. The clinical presentation was that of a severe pneumonitis while lung biopsy revealed diffuse alveolar damage. Cardiac symptoms and changes in blood indices in this patient support the possible association between both and PM exposure suggested by epidemiological studies.