Biochemical assays in lung homogenates: Artifacts caused by trapped blood after perfusion

Cross, Carroll E.; Watanabe, Tyrus T.; Hasegawa, Glen K.; Goralnik, Gary N.; Roertgen, K. E.; Kaizu, Tokio; Reiser, Karen M.; Gorin, Arnold B.

doi:10.1016/s0041-008x(79)80012-5

Cited by 49 publications

(19 citation statements)

References 33 publications

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“…Aliquots of this blood were diluted 1: 1000 with 0.1 M Tris-HC1, 1% Triton X-100, pH 7.0, and sonicated for 15 s at 4°C. The absorbance of 1 ml of fetal blood lysate was recorded from 350 to 650 nm before and after the addition of 100 pl of 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, Fullerton, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Aliquots (5,10,20,30, or 40 pl) of the 1: 1000 blood sonicate were added to 100 pl lung homogenate for each litter and made up to 1000-p1 final volume with 0.1 M Tris-HC1, 1 % Triton X-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 .…”

Section: Methodsmentioning

confidence: 99%

“…Reported values have been corrected for enzyme activity contributed by contaminating blood by a modification of the method described by Cross et al (5), using homologous blood supplements.…”

mentioning

confidence: 99%

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

Tanswell

1984

Pediatr Res

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SummaryIt 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-6-phosphate dehydrogenase, were unaffected by maternal iron supplements. AbbreviationsCuZn SOD, copper zinc superoxide dismutase CAT, catalase G-6-PD, glucose-6-phosphate dehydrogenase GPx, glutathione peroxidase Mn SOD, manganese superoxide dismutase HBSS, Hanks' balanced salt solution Undernutrition is reported to enhance pulmonary oxygen toxicity (10). One potential contributing factor is a loss of metalloprotein antioxidant enzymes activities upon metal depletion. This has been reported with copper (4) for CuZn SOD, with manganese (6) for Mn SOD, with iron and selenium (18) for GPx, and with iron (20) for CAT.We have previously reported that the specific activity of lung catalase of gravid female rats fed Purina Lab Chow ad libitum declined with gestation (13). The fetuses of these animals demonstrated gestation-dependent increases in specific activity of CuZn SOD, Mn SOD, GPx, and G-6-PD in lung between day 18 and day 22 (term) of gestation (24). The specific activity of fetal lung CAT, however, increased between days 18 and 20 of gestation, but showed no significant increase between day 20 and term (24). Since the fall in maternal lung CAT activity towards the end of gestation was reversed by parenteral iron supplements, it is possible that the blunted increase in fetal lung catalase reflected limited iron transport across the placenta.In this report, we examine the effect of maternal iron supplementation on fetal lung growth, and on the specific activity of oxygen-protective enzymes of fetal rats sacrificed prematurely on day 2 1 of gestation. Reported values have been corrected for Received June 13, 1983;accepted February 7, 1984 enzyme activity contributed by contaminating blood by a modification of the method described by Cross et al. (5), using homologous blood supplements. MATERIALS AND METHODSFemale 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 decapitated in utero to prevent respiration and immediately removed from the uterus. Each fetus was weighed and the carcasses were placed in HBSS at 4°C. The chest cage was opened from diaphragm to neck; then the heart and lungs were removed en bloc and...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Tanswell

1984

Pediatr Res

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“…The supernatant was used for antioxidant enzyme assays and DNA content. Hb concentrations were determined in lung homogenates and blood and used to correct the amount of '311-albumin (19) and antioxidant enzyme activities (20) contributed by intravascular blood.…”

Section: Methodsmentioning

confidence: 99%

Corticosteroids, Thyrotropin-Releasing Hormone, and Antioxidant Enzymes in Preterm Lamb Lungs

et al. 1991

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ABSTRACT. Forty-three twin lamb fetuses of 121 f: 1 d gestation were catheterized and received i.v. saline (n = 8), 0.75 mg/kg/h cortisol for 60 h (n = IS), 5 ~g / k g thyrotropin-releasing hormone (TRH) every 12 h for five doses (n = 9), or cortisol and TRH (n = 11) before delivery at 128 + 1 d. After delivery, the lambs were randomized for natural sheep surfactant treatment or sham treatment, ventilated for 75 min, and killed. Superoxide dismutase, catalase, and glutathione peroxidase activities were measured in fetal lung tissue. Superoxide dismutase and catalase activities were increased in both the corticosteroid (p < 0.001) and the corticosteroid with TRH ( p < 0.01) groups.Glutathione peroxidase activity was higher after prenatal corticosteroid treatment, but statistical significance was not reached ( p = 0.06). Although prenatal exposure to corticosteroids increased superoxide dismutase, catalase, and glutathione peroxidase activities, TRH alone or TRH added to corticosteroids provided no additional benefit. Lambs treated with surfactant had higher lung catalase activities than lambs that did not receive surfactant, probably secondary to the presence of catalase activity in the surfactant preparation. Increased pulmonary antioxidant enzyme activity may be an additional feature of the overall beneficial effect of corticosteroids on fetal lung development. (Pediatr Res 30: 518-521, 1991) Abbreviations GPX, glutathione peroxidase SOD, superoxide dismutase T3, triiodothyronine T4, thyroxine ~~~, -t h~r o t r o~i n -r e l e a s i n~ hormone VEI, ventilatory efficiency index Exposure of the lung to high concentrations of oxygen produces endothelial and alveolar type I cell damage associated with alveolar-capillary leakage, lung edema, and, ultimately, deterioration of lung function. Alveolar type I1 cells synthesize and secrete surfactant and maintain the integrity of the alveolar epithelium by proliferation and differentiation into type I cells

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“…T o measure lung blood content, we modified the method described by Cross ct ul. (16) for use on the microtiter plate reader. First, we incubated 25 pL of sample with 175 pL of a solution of 1 % Triton in 0.1 M Tris maleate (pH 7.0) for 5 min and measured the O D at 410 nm.…”

Section: Gr Glutathione Reductasementioning

confidence: 99%

Endotoxin Induces Glutathione Reductase Activity in Lungs of Mice

et al. 1994

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ABSTRACT. Glutathione reductase catalyzes the NADPH-dependent conversion of glutathione disulfide to glutathione and helps protect the lung from injury by reactive oxygen. In animals allowed to breathe nearly 100% oxygen, the activities of other antioxidants in the lung can be induced by treatment with endotoxin, and this induction is associated with increased tolerance to hyperoxia. The purpose of this study was to see whether glutathione reductase activity in the lungs of mice increased with endotoxin treatment alone. We studied 60 FVB mice (20 males and 40 females). Half received endotoxin (500 pglkg) intraperitoneally at time 0 and 24 h, and the controls received an equal volume of saline. At 48 h we killed the mice and removed their lungs. Treatment of mice with endotoxin increased glutathione reductase activity in the lung 55% (0.035 f 0.005 to 0.054 f 0.010 pmol NADPH reduced/min/mg protein; mean f SD; endotoxin different from control, p < 0.001). The increase in activity was the same for male and female mice. We measured the specific protein for glutathione reductase by Western analysis and mRNA for glutathione reductase using a slot-blot analysis and found that both increased roughly 2-fold with endotoxin treatment. This suggests that endotoxin treatment resulted in either increased rate of transcription of glutathione reductase mRNA or increased mRNA stability. We conclude that endotoxin treatment increases glutathione reductase activity in the lung and that this increase in activity may play a role in subsequent protection from hyperoxia. (Pediatr Res 35: 311-315, 1994) Abbreviations is GSH. In a reaction catalyzed by the selenium-dependent and selenium-independent glutathione peroxidases, hydrogen peroxide and lipid hydroperoxides are reduced to water or their respective alcohols, whereas GSH is oxidized to GSSG. GSSG is subsequently reduced to GSH at the expense of NADPH oxidation in a reaction catalyzed by GR. The importance of GSH in protecting cells from reactive oxygen has been underscored by experiments showing that depletion of intracellular GSH ( 1 , 2) or inhibition ofglutathione peroxidase (3) or G R (4) significantly augments the toxic effects of oxygen on the lung.Previously, we found that GR activity decreased more than 50% in the lungs of lambs dying of oxygen toxicity and that this decrease was associated with a near doubling of GSSG in their lung tissue (5). More recently, we showed that plasma concentrations of GSSG are increased in premature infants compared with adults, suggesting that this population may be deficient in G R (6). If reduced GR activity is playing a role in oxygen-induced lung injury, then increasing intracellular GR activity might help protect the lung from oxygen toxicity. The purpose of our experiments was to attempt to increase GR activity in the lungs of mice by treating the mice with endotoxin and to determine whether this increase in activity was associated with increased steady state concentrations of mRNA for GR. We chose endotoxin for these experiments ...

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Biochemical assays in lung homogenates: Artifacts caused by trapped blood after perfusion

Cited by 49 publications

References 33 publications

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

Corticosteroids, Thyrotropin-Releasing Hormone, and Antioxidant Enzymes in Preterm Lamb Lungs

Endotoxin Induces Glutathione Reductase Activity in Lungs of Mice

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