Glutathione Localization and Distribution after Intratracheal Instillation Implications for Treatment1-3
LEWIS J. SMITH, JAMES ANDERSON, and MIR SHAMSUDDIN
Introduction SUMMARY To gain insight into how glutathione given directly into the lung protects fasted mice
Exposure to high concentrations of oxagainst hyperoxic lung damage and to provide a framework for developing treatment strategies in ygen produces lung damage in severalanpatients, we determined the lung distribution and retention of Intratracheally administered glutathione imal species, including humans (1). Fast(GSH) and Its fate after leaving the lung. Mice received an intratracheal injection of ['HIGSH with ing increases the susceptibility to oxygenor without cold GSH and with or without IIposomes. The distribution of the 'H label was equal In induced lung damage (2). This appears both lungs, but the left lung had a higher concentration because of Its smaller size. The 'H label was cleared rapidly from the lung: only 1% remained at 24 h, and more than 50% of the label at to be due to, at least in part, a reduction that time was no longer attached to the GSH. Administration of GSH with Iiposomes Increased the in lung glutathione (aSH) levels and the retention of GSH by 20 to 50%, but the amount remaining at 24 h was still only 1%. The increase inability to increase aSH and the enzyme associated with the Iiposomes was due to enhanced retention of the GSH encapsulated in the Iipoglutathione reductase (Gk) in response somes, not the much larger amount present free in the GSH·llposome mixture. Fasting and exposure to the oxidant stress (3, 4). Intratracheal to 100% oxygen had little effect on GSH retention. Some of the 'H label leaving the lung was excreted administration of aSH with liposomes, by the kidneys, a small amount was retained In the liver, and a large amount accumulated In the but not aSH alone, to oxygen-exposed blood. Of the amount in the blood, about 60% was in red blood cells (RBC) and the rest in plasma. fasted mice delaysthe onset and decreases Much of the 'H label in RBC and lung at 24 h was no longer attached to the GSH, whereas most the severity of the lung damage (3). Lipoin the plasma and liver was. These results suggest that the beneficial effects of GSHgiven to oxygensomes given without aSH or other anexposed fasted mice are not due to the administered GSH alone. The Iiposome carrier, through one or more mechanisms, may play an Important role. These factors should be considered before tioxidants also have a beneficial effect Initiating human studies designed to increase lung levels of GSH and possibly other antioxidants. in this model (3) and in a rat model of AM REV RESPIR DIS 1992; 145:153-159 hyperoxic lung damage (5, 6). In addition to animal studies exploring the possible beneficial effects of supplemental aSH in the setting of oxidantinduced lung damage, there has been re- fects achieved by the intratracheal ad- carbon dioxide concentration below 0.5%. cent interest in providing aSH to patients ministration of aSH plus liposomes in Oxygen and carbon dioxide concentrations with lung disease. This is a consequence fasted mice exposed to 1000/0 oxygen are were monitored with Beckman OM-ll and of recent reports describing reduced al- not due to increased lung concentrations LB-2 analyzers (Beckman Instruments, Fullerton, CA). The temperature was maintained veolar epithelial lining fluid levels of of aSH resulting from administration of between 23.9° and 26.7° C and the relative aSH in patients with HIV infection (7) the aSH component of the aSH-lipo- humidity between 40 and 50%. and in patients with interstitial lung dis- some mixture. Studies designed to inMice were fed a normal diet (fed) or had ease (8). However, there are limited data crease aSH concentrations in the lung all food removed (fasted). Water was providfrom either animal (3, 9) or human (10) must consider its rapid lung clearance ed to all mice. Mice received intratracheal instudies on the fate of aSH administered when given alone and when given with jections of either [3HjGSH alone, ['HjGSH + cold GSH, or [3HjGSH + cold GSH + liposomes. directly onto the lung. liposomes, as noted below. The [3HjGSH (250 To better understand how direct adMethods ministration of aSH into the lung proExperimental Animals and tects fasted mice from the damage pro(Received in originalform December 28, 1990and Lung Injury in revised form July 24, 1991) duced by exposure to high concentrations of oxygen, and to provide a framework Male Balb-c mice (Charles River, Portage, From the Department of Medicine (Pulmonary for developing and testing treatment MI), 8 to 10 wk of age and weighing 20 to Section), Northwestern Universityand Veterans Adstrategies in patients, we determined the 25 g, were housed 10 mice to a cage. For the ministration Lakeside Medical Center, Chicago, distribution and retention in the lung of GSH distribution and retention studies, they Illinois. were maintained in room air under controlled Supported by the Veterans Administration Reintratracheally administered aSH and its environmental conditions. For the oxygen exfate after leaving the lung. We found that posure experiments, they were placed in poly- search Service and the Bazley Foundation. 3 Correspondence and requests for reprints aSH given intratracheally is cleared rap- ethylene "glove bags" (Baxter Healthcorp, should be addressed to Lewis J. Smith, M.D., Pulidly from the lung and increases the lung McGaw Park, IL), and exposed to either monary Section, Northwestern University MediaSH content only transiently. These 100070 oxygen (98 ± 1%) or air for as long cal Center, Wesley 456, 250 E. Superior, Chicago, findings suggest that the beneficial ef- as 4 days. Flow rates were set to maintain the IL 60611. 1
2
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SMITH, ANDERSON, AND SHAMSUDDIN
mCi/mmol; New England Nuclear, Boston, MA) was given as a dose of approximately 1 x lOs cpm in a OJ-ml volume unless otherwise noted. For the autoradiographic and GSH separation studies 1 x 106 cpm were given in the same volume. The amount of cold GSH administered was either 1,000 or 2,500 J.Lg (0.1 ml of a 10- or 25-mg/ml solution). For all experiments requiring intratracheal injections, mice were first anesthetized with pentobarbital, 30 mg/kg, given intraperitoneally. An incision was made in the neck, the trachea was exposed, and a 27-gauge needle was inserted into the trachea to instill 0.1 ml of the appropriate material. The mice were elevated 30 degrees above the horizontal plane during the instillation procedure and maintained in that position until they recovered from the anesthesia. Experiments were terminated by anesthetizing the mice with a higher dose of pentobarbital, 50 mg/kg, The lungs were perfused via the right ventricle at 20 em H 2 0 pressure with cold isotonic saline until they became white. They were removed from the chest, trimmed of extraneous tissue, including trachea and main bronchi, weighed, and homogenized (see below). In the experiments in which bronchoalveolar lavage was also performed, the lungs were perfused with saline, the trachea was cannulated with a no. 18 x 1.25-inch plastic intravenous catheter filledwith phosphate-buffered saline (PBS), and the lungs were lavaged four times with separate 1-ml aliquots of cold PBS. The bronchoalveolar lavage (BAL) effluent, > 90
LEWIS J. SMITH, JAMES ANDERSON, and MIR SHAMSUDDIN
Introduction SUMMARY To gain insight into how glutathione given directly into the lung protects fasted mice
Exposure to high concentrations of oxagainst hyperoxic lung damage and to provide a framework for developing treatment strategies in ygen produces lung damage in severalanpatients, we determined the lung distribution and retention of Intratracheally administered glutathione imal species, including humans (1). Fast(GSH) and Its fate after leaving the lung. Mice received an intratracheal injection of ['HIGSH with ing increases the susceptibility to oxygenor without cold GSH and with or without IIposomes. The distribution of the 'H label was equal In induced lung damage (2). This appears both lungs, but the left lung had a higher concentration because of Its smaller size. The 'H label was cleared rapidly from the lung: only 1% remained at 24 h, and more than 50% of the label at to be due to, at least in part, a reduction that time was no longer attached to the GSH. Administration of GSH with Iiposomes Increased the in lung glutathione (aSH) levels and the retention of GSH by 20 to 50%, but the amount remaining at 24 h was still only 1%. The increase inability to increase aSH and the enzyme associated with the Iiposomes was due to enhanced retention of the GSH encapsulated in the Iipoglutathione reductase (Gk) in response somes, not the much larger amount present free in the GSH·llposome mixture. Fasting and exposure to the oxidant stress (3, 4). Intratracheal to 100% oxygen had little effect on GSH retention. Some of the 'H label leaving the lung was excreted administration of aSH with liposomes, by the kidneys, a small amount was retained In the liver, and a large amount accumulated In the but not aSH alone, to oxygen-exposed blood. Of the amount in the blood, about 60% was in red blood cells (RBC) and the rest in plasma. fasted mice delaysthe onset and decreases Much of the 'H label in RBC and lung at 24 h was no longer attached to the GSH, whereas most the severity of the lung damage (3). Lipoin the plasma and liver was. These results suggest that the beneficial effects of GSHgiven to oxygensomes given without aSH or other anexposed fasted mice are not due to the administered GSH alone. The Iiposome carrier, through one or more mechanisms, may play an Important role. These factors should be considered before tioxidants also have a beneficial effect Initiating human studies designed to increase lung levels of GSH and possibly other antioxidants. in this model (3) and in a rat model of AM REV RESPIR DIS 1992; 145:153-159 hyperoxic lung damage (5, 6). In addition to animal studies exploring the possible beneficial effects of supplemental aSH in the setting of oxidantinduced lung damage, there has been re- fects achieved by the intratracheal ad- carbon dioxide concentration below 0.5%. cent interest in providing aSH to patients ministration of aSH plus liposomes in Oxygen and carbon dioxide concentrations with lung disease. This is a consequence fasted mice exposed to 1000/0 oxygen are were monitored with Beckman OM-ll and of recent reports describing reduced al- not due to increased lung concentrations LB-2 analyzers (Beckman Instruments, Fullerton, CA). The temperature was maintained veolar epithelial lining fluid levels of of aSH resulting from administration of between 23.9° and 26.7° C and the relative aSH in patients with HIV infection (7) the aSH component of the aSH-lipo- humidity between 40 and 50%. and in patients with interstitial lung dis- some mixture. Studies designed to inMice were fed a normal diet (fed) or had ease (8). However, there are limited data crease aSH concentrations in the lung all food removed (fasted). Water was providfrom either animal (3, 9) or human (10) must consider its rapid lung clearance ed to all mice. Mice received intratracheal instudies on the fate of aSH administered when given alone and when given with jections of either [3HjGSH alone, ['HjGSH + cold GSH, or [3HjGSH + cold GSH + liposomes. directly onto the lung. liposomes, as noted below. The [3HjGSH (250 To better understand how direct adMethods ministration of aSH into the lung proExperimental Animals and tects fasted mice from the damage pro(Received in originalform December 28, 1990and Lung Injury in revised form July 24, 1991) duced by exposure to high concentrations of oxygen, and to provide a framework Male Balb-c mice (Charles River, Portage, From the Department of Medicine (Pulmonary for developing and testing treatment MI), 8 to 10 wk of age and weighing 20 to Section), Northwestern Universityand Veterans Adstrategies in patients, we determined the 25 g, were housed 10 mice to a cage. For the ministration Lakeside Medical Center, Chicago, distribution and retention in the lung of GSH distribution and retention studies, they Illinois. were maintained in room air under controlled Supported by the Veterans Administration Reintratracheally administered aSH and its environmental conditions. For the oxygen exfate after leaving the lung. We found that posure experiments, they were placed in poly- search Service and the Bazley Foundation. 3 Correspondence and requests for reprints aSH given intratracheally is cleared rap- ethylene "glove bags" (Baxter Healthcorp, should be addressed to Lewis J. Smith, M.D., Pulidly from the lung and increases the lung McGaw Park, IL), and exposed to either monary Section, Northwestern University MediaSH content only transiently. These 100070 oxygen (98 ± 1%) or air for as long cal Center, Wesley 456, 250 E. Superior, Chicago, findings suggest that the beneficial ef- as 4 days. Flow rates were set to maintain the IL 60611. 1
2
153
154
SMITH, ANDERSON, AND SHAMSUDDIN
mCi/mmol; New England Nuclear, Boston, MA) was given as a dose of approximately 1 x lOs cpm in a OJ-ml volume unless otherwise noted. For the autoradiographic and GSH separation studies 1 x 106 cpm were given in the same volume. The amount of cold GSH administered was either 1,000 or 2,500 J.Lg (0.1 ml of a 10- or 25-mg/ml solution). For all experiments requiring intratracheal injections, mice were first anesthetized with pentobarbital, 30 mg/kg, given intraperitoneally. An incision was made in the neck, the trachea was exposed, and a 27-gauge needle was inserted into the trachea to instill 0.1 ml of the appropriate material. The mice were elevated 30 degrees above the horizontal plane during the instillation procedure and maintained in that position until they recovered from the anesthesia. Experiments were terminated by anesthetizing the mice with a higher dose of pentobarbital, 50 mg/kg, The lungs were perfused via the right ventricle at 20 em H 2 0 pressure with cold isotonic saline until they became white. They were removed from the chest, trimmed of extraneous tissue, including trachea and main bronchi, weighed, and homogenized (see below). In the experiments in which bronchoalveolar lavage was also performed, the lungs were perfused with saline, the trachea was cannulated with a no. 18 x 1.25-inch plastic intravenous catheter filledwith phosphate-buffered saline (PBS), and the lungs were lavaged four times with separate 1-ml aliquots of cold PBS. The bronchoalveolar lavage (BAL) effluent, > 90
