0031-3998/01/4902-0181 PEDIATRIC RESEARCH Vol. 49, No. 2, 2001 Copyright © 2001 International Pediatric Research Foundation, Inc. Printed in U.S.A. Superoxide Dismutase and Catalase Do Not Affect the Pulmonary Hypertensive Response to Group B Streptococcus in the Lamb DAVID T. CARPENTER, HEATHER R. LARKIN, AUDREY S. CHANG, ELENA MORRIS, J. TIMOTHY O’NEILL, AND JERRI CURTIS Uniformed Services University of the Health Sciences, Department of Pediatrics, Bethesda, MD 20814-4799 [D.T.C., H.R.L., T.O., J.C.]; Walter Reed Army Medical Center, Department of Clinical Investigation [A.S.C], Department of Biochemistry [E.M.], Washington, D.C. 20307-5001, U.S.A.; National Naval Medical Center, Department of Pediatrics, Bethesda, MD 20889-5600, U.S.A. [J.C.] ABSTRACT The purpose of this study was to determine whether treatment ninefold increase in plasma enzyme activities. As opposed to with conjugated antioxidant enzymes could attenuate or abolish previously published data from endotoxin models, PEG-CAT pulmonary hypertension induced by group B streptococcus and PEG-SOD were ineffective in altering the GBS-induced (GBS). Lambs, 3–7 d old, were anesthetized and ventilated. pulmonary hypertensive response in this model. This suggests Intravascular catheters were placed in the left ventricle, descend- that acute administration of antioxidant enzymes may not be ing aorta, right atrium, and pulmonary artery for continuous effective in ameliorating GBS-induced pulmonary hypertension. monitoring of intravascular pressures. Cardiac output was mea- (Pediatr Res 49: 181–188, 2001) sured with radiolabeled microspheres. Measurements were ob- tained at baseline and 15 and 60 min into a 60-min GBS infusion, Abbreviations: and 60 min after GBS was stopped. Blood gas values were held CAT, catalase Ͼ constant and Pao2 was maintained 100 mm Hg. The control CI, cardiac index (mL/kg/min) group received saline vehicle only (n ϭ 6), the GBS group ENZ, enzymes received GBS infusion only (n ϭ 9), the enzymes (ENZ) group GBS, group B streptococcus (Streptococcus agalactiae) received polyethylene glycol-superoxide dismutase (PEG-SOD) MAP, mean systemic arterial pressure (mm Hg) and polyethylene glycol-catalase (PEG-CAT) treatment only (n MPAP, mean pulmonary arterial pressure (mm Hg) ϭ 6), and the ENZϩGBS group received PEG-SOD and PEG- PEG, monomethoxy-polyethylene glycol CAT then GBS (n ϭ 9). Plasma samples were obtained to PEG-CAT, polyethylene glycol-catalase confirm increased superoxide dismutase and catalase activities in PEG-SOD, polyethylene glycol-superoxide dismutase the groups receiving enzymes. Compared with baseline, pulmo- PVRI, pulmonary vascular resistance index (mm nary vascular resistance increased by 119% and 101% at 15 min Hg⅐mL-1⅐kg-1⅐min-1) and 87% and 81% at 60 min in the GBS and ENZϩGBS groups, ROS, reactive oxygen species respectively. Sixty minutes after the termination of the GBS SOD, superoxide dismutase infusion, PVR returned to baseline in the GBS group but did not SVRI, systemic vascular resistance index (mm in the ENZϩGBS group. Enzyme infusions resulted in at least a Hg⅐mL-1⅐kg-1⅐min-1) Morbidity and mortality remain unacceptably high in of antibiotics, both intrapartum and immediately following newborn infants infected with GBS despite aggressive use delivery (1). Many infants infected with this organism de- velop pulmonary hypertension (2), even in the face of blood cultures that have quickly become sterile. In many of these Received October 1, 1999; accepted October 13, 2000. Correspondence and reprint requests: Jerri Curtis, CDR, M.C., U.S.N., Uniformed infants, the high morbidity and mortality is a result of the Services University of the Health Sciences, Department of Pediatrics; Room C1066, 4301 pulmonary hypertension. There are no unique or specific Jones Bridge Road, Bethesda, Maryland 20814-4799, U.S.A. Supported by funds from The Chief, Navy Bureau of Medicine and Surgery, Wash- ington, D.C., Clinical Investigation Program sponsored this study #B97-077; Uniformed Services University of the Health Sciences #R086CT; Walter Reed Army Medical Center, The research reported herein was conducted according to the principles set forth in the Department of Clinical Investigation WU #6415. Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Re- The views expressed in this article are those of the authors and do not reflect the official sources, National Research Council, HHS, Pub. No. (NIH) 85-23, revised 1985. policy or position of the Department of the Navy, Department of the Army, Department Presented in part at the American Academy of Pediatrics Fall Meeting, October 1998; of Defense, nor the U.S. Government. San Francisco, CA, U.S.A. 181 182 CARPENTER ET AL. therapies available to reverse this abnormal pathophysio- METHODS logic process once elicited by the group B streptococcal organism. Outcomes have improved over the past decade Animal preparation. Mixed-strain newborn lambs (3–7 d of with the use of extracorporeal membrane oxygenation, in- age) were anesthetized with i.v. sodium pentobarbital (induc- -1 -1 haled nitric oxide, and more sophisticated high-frequency tion dose, 30 mg/kg; maintenance, 5–7 mg⅐kg ⅐h ). The tra- ventilators. However, these treatment modalities are not chea was intubated and animals were ventilated using a Har- specific to this disease process. There is an increasing body vard Model 665 animal volume ventilator (Harvard Apparatus of literature suggesting that ROS play a significant role in Co., South Natick, MA, U.S.A.). Continuous noninvasive GBS-induced pulmonary hypertension. Early scavenging of monitoring of end-tidal Co2, Fio2, and O2 saturation was ROS may offer a specific therapeutic treatment regimen in attained via a BCI Model 9100 multi gas monitor (BCI Inter- those infants who have this diagnosis. This could dramati- national, Waukesha, WI, U.S.A.) and Ohmeda 4700 pulse cally improve their outcomes. oximeter (Ohmeda Corp., Louisville, CO, U.S.A.). Ventilator settings and supplemental oxygen were adjusted to maintain an When pulmonary intravascular macrophages engulf bacte- arterial Pco of 4.7–6.0 kPa (35–45 mm Hg) and a Po of ria, ROS are produced (3). ROS are essential for bacterial 2 2 Ͼ13.3 kPa (Ͼ100 mm Hg). Arterial pH was maintained be- killing, however, they may be a double-edged sword. Several tween 7.35–7.45, with sodium bicarbonate used as necessary. investigators have proposed that ROS are early mediators of Rectal temperature was servo-controlled (YSI model 73A, pulmonary hypertension (4–6). For example, pulmonary hy- Yellow Springs Instruments, Yellow Springs, OH, U.S.A.) and pertension induced by endotoxin in the sheep is attenuated by maintained at 39–40°C via a heating blanket and heat lamp catalase, indicating that hydrogen peroxide may participate in wired to a rectal probe. Arterial blood gas values and pH are the generation of increased pulmonary arterial pressures (6, 7). reported at the animals’ body temperature. However, other studies have resulted in inconsistent responses Catheters were placed in the pulmonary artery, left ventricle, (8–11). Olson et al. (12) hypothesized that the short half-life of right atrium, subclavian artery, femoral artery, and femoral antioxidant enzymes was responsible for the variability in vein through peripheral cutdown sites. Pressure transducers responses. They attempted to overcome this problem by using (Statham Db-23 Gould Instrument Systems, Cleveland, OH, antioxidant enzymes that had been conjugated to PEG, which U.S.A.) were used for continuous recording of mean and greatly increased the half-life. They used SOD or CAT bound phasic pressures on a polygraphic recorder (Gould eight- to PEG in a pig model of endotoxin-induced pulmonary hy- channel strip-chart recorder, model 2017-8868-005180, Cleve- pertension. PEG-SOD alone did not offer any protection, but land, OH, U.S.A.). Left ventricular and pulmonary arterial PEG-CAT significantly blunted the endotoxin-induced pulmo- catheters were placed by observing characteristic pressure nary injury. A combination of the PEG-bound enzymes was waveforms and confirmed in all study animals during the not studied. necropsy. Following catheter placement, each animal was par- Other investigators have examined the role of ROS in the alyzed with 0.10 mg/kg of pancuronium bromide and antico- development of GBS-induced pulmonary hypertension using agulated with 500 U/kg of heparin to maintain line patency. dimethylthiourea as a hydroxyl radical scavenger (4, 13–15). There was a 30-min postoperative stabilization period before All have demonstrated significant attenuation of GBS-induced the start of the experimental protocol. rises in pulmonary artery pressure or pulmonary vascular Heat-killed GBS preparation. Type III GBS (strain 893) resistance in the animals pretreated with dimethylthiourea. were plated and incubated at 37°C overnight on blood agar. However, there have been doubts regarding the specificity that Pure colonies were transferred to Todd-Hewett broth and dimethylthiourea has for hydroxyl radical scavenging (16). grown in a 37°C water bath to log phase. This bacterial suspension was centrifuged and washed twice with sterile Therefore, the question regarding the role of ROS in the normal saline to remove broth and then heat-killed in a 60°C mechanism of GBS-induced pulmonary hypertension remains water bath for 1 h. The suspension was then diluted with sterile unanswered. normal saline to an OD of 0.6 (1.0 ϫ 1012 colony forming units The aim of the present study was to determine whether per liter). The final bacterial suspension was cultured to con- early treatment with both conjugated antioxidant enzymes firm sterility. (PEG-SOD and PEG-CAT) could attenuate or abolish pul- Antioxidant enzyme preparation. PEG-SOD (EC 1.15.1.1) monary hypertension induced by GBS. We hypothesized and PEG-CAT (EC 1.11.1.6) were purchased from Oxis (Port- that pulmonary intravascular macrophages are activated by land, OR, U.S.A.).