Pulmonary Antioxidant Enzyme Maturation in the Fetal and Neonatal Rat. II. The Influence of Maternal Iron Supplements upon Fetal Lung Catalase Activity

PULMONARY ANTIOXIDANT ENZYME MATURATION 003 1-3998/84/1809-087 1$02.00/0 PEDIATRIC RESEARCH Copyright O 1984 International Pediatric Research Foundation, Inc. Vol. 18, No. 9, 1984 Printed in U.S.A. Pulmonary Antioxidant Enzyme Maturation in the Fetal and Neonatal Rat. 11. The Influence of Maternal Iron Supplements upon Fetal Lung Catalase Activity A. KEITH TANSWELL AND BRUCE A. FREEMAN Department of Paediatrics, University of Western Ontario, London, Canada and Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710 Summary It was obsegved that the lung catalase activity of premature (day 21 of gestation; term = 22 days) rat pups is affected by maternal iron intake. Pups from control dams receiving Purina Lab Chow and water ad libitum have only 50% of the lung catalase activity of pups from dams who received 1 mg/kg parenteral iron dextran daily from day 7 to day 20 of gestation. Other oxygen-protective enzymes, copper-zinc and manganese superoxide dismutase, glutathione peroxidase, and glucose-6phosphate dehydrogenase, were unaffected by maternal iron supplements. enzyme activity contributed by contaminating blood by a modification of the method described by Cross et al. (5), using homologous blood supplements. MATERIALS AND METHODS Female Sprague-Dawley white rats of 275-300 g (Charles River Inc., St. Constant, Quebec) were mated with male hooded rats and delivered to our animal quarters the following day. From day 7 to day 20 of gestation, the gravid rats were injected intramuscularly with either 1 mg/kg/day of iron dextran in isotonic saline or isotonic saline alone, or received no injections. On day 21 of gestation (term = 22 days), the animals were sacrificed by excess chloroform anesthesia; the fetuses were deAbbreviations capitated in utero to prevent respiration and immediately reCuZn SOD, copper zinc superoxide dismutase moved from the uterus. Each fetus was weighed and the carcasses CAT, catalase were placed in HBSS at 4°C. The chest cage was opened from G-6-PD, glucose-6-phosphate dehydrogenase diaphragm to neck; then the heart and lungs were removed en GPx, glutathione peroxidase bloc and kept in HBSS at 4°C. The heart and major airways were Mn SOD, manganese superoxide dismutase dissected off and the lungs from each fetus were washed with HBSS, Hanks' balanced salt solution HBSS, blotted dry, and weighed. The lungs from each litter were then pooled, minced, and homogenized in 5 ml 50 mM potasUndernutrition is reported to enhance pulmonary oxygen sium phosphate, 0.1 mM EDTA, pH 7.8 for 30 s at 4°C with a toxicity (10). One potential contributing factor is a loss of me- Polytron (Brinkmann Instruments, Inc., Westbury, NY). The talloprotein antioxidant enzymes activities upon metal depletion. homogenates were centrifuged at 1000 x g x 10 min to remove This has been reported with copper (4) for CuZn SOD, with fibrous material and the supernatants were stored at -70°C prior manganese (6) for Mn SOD, with iron and selenium (18) for to analysis. Preliminary observations showed no loss of enzyme activity with centrifugation or a single freeze-thaw of homogeGPx, and with iron (20) for CAT. We have previously reported that the specific activity of lung nates. Protein content was estimated by the Lowry method (19), and catalase of gravid female rats fed Purina Lab Chow ad libitum DNA was estimated fluorimetrically (7). CAT (EC 1.1 1.1.6.) was declined with gestation (13). The fetuses of these animals demonstrated gestation-dependent increases in specific activity of measured as described by Bergmeyer (2), GPx (EC 1.1 1.1.9.) as CuZn SOD, Mn SOD, GPx, and G-6-PD in lung between day described by Beutler (3), and G-6-PD (EC 1.1.1.49.), CuZn SOD, 18 and day 22 (term) of gestation (24). The specific activity of and Mn SOD (EC 1.15.1.1.) as described by Hayatdavoudi et al. fetal lung CAT, however, increased between days 18 and 20 of (14). A number of homogenates from each group also had CAT estimations performed in the presence of to loT6M iron as gestation, but showed no significant increase between day 20 and femc nitrate (Fe(N03)3. 9H20). term (24). Since the fall in maternal lung CAT activity towards A separate group of day 2 1 fetuses were sacrificed and decapthe end of gestation was reversed by parenteral iron supplements, itated, and blood was collected from the neck vessels by gravity it is possible that the blunted increase in fetal lung catalase drainage. Aliquots of this blood were diluted 1:1000 with 0.1 M reflected limited iron transport across the placenta. In this report, we examine the effect of maternal iron supple- Tris-HC1, 1% Triton X-100, pH 7.0, and sonicated for 15 s at mentation on fetal lung growth, and on the specific activity of 4°C. The absorbance of 1 ml of fetal blood lysate was recorded oxygen-protective enzymes of fetal rats sacrificed prematurely from 350 to 650 nm before and after the addition of 100 pl of on day 2 1 of gestation. Reported values have been corrected for 0.1 M NaOH, 0.1 M Na2S204,as described by Cross et al. (5) in a Gilford 252 spectrophotometer (Gilford Instruments, Oberlin, OH) with a Beckman DU optical system (Beckman Instruments, Received June 13, 1983; accepted February 7, 1984. Fullerton, CA). Address correspondence to Dr. A. K. Tanswell, Research Institute, St. Joseph's Aliquots (5, 10, 20, 30, or 40 pl) of the 1:1000 blood sonicate Hospital, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada. were added t o 100 pl lung homogenate for each litter and made Supported by grants MA 7867 from the Medical Research Council of Canada up to 1000-p1 final volume with 0.1 M Tris-HC1, 1% Triton X(A. K. T.), and HL-29784 from the National Institutes of Health (B. A. F.). 872 TANSWELL AND FREEMAN 100, pH 7.0, sonicated for 15 s on ice, and then centrifuged at 11,000 x g for 10 min. The "dithionite index" for each sample was calculated as described by Cross et al. (5), except that the wavelength used was 405 nm, by the formula: dithionite index =A405- (1.1 X DTA405)where DTA,, represents the absorbance in the presence of 100 4 0 . 1 M NaOH, 0. l M Na2S204and the factor of 1.1 corrects for dilution. The dithionite index obtained by addition of blood sonicate was plotted for each sample and found to be indicative of successively increasing blood contamination. From this, the original blood contamination in the sample could be calculated by extrapolation from blood-contaminated dithionite index plots. Aliquots of blood were also examined for CAT, CuZn SOD, Mn SOD, G-6-PD, and GPx activity and, from the previous calculations of blood contamination, it was possible to correct each lung enzyme specific activity for enzyme activity due to blood contamination. All results are presented as means + SEM unless otherwise stated, and statistical comparisons were made by analysis of x P -I 4 ADDED BLOOD Fig. 2. Measurement of lung homogenate blood contamination. The line connecting the dithionite (...truncated)