Charles V. Smith

Oxidant stress responses in premature infants during exposure to hyperoxia.

ABSTRACT: To assess oxidant stress responses in newborn infants treated with elevated concentrations of oxygen, we measured plasma concentrations of glutathione (GSH) and glutathione disulfide (CSSG) in newborn infants ranging from 23 to 42 wk gestational age. All infants recruited into the study were mechanically ventilated and had catheters placed in their umbilical arteries as part of their normal clinical management. Blood samples were obtained on d 1, 3, and 5 and weekly thereafter or until the catheters were removed. We observed plasma concentrations of GSSG in these infants that were frequently an order of magnitude higher than the 0.1 to 0.3 μM we find in adults. Interestingly, plasma GSSG concentrations were inversely correlated to the inspired oxygen tensions. This effect appeared to arise from the patient selection criteria whereby, of the infants studied, those breathing the lowest partial pressures of oxygen were the smallest and gestationally youngest. A second observation was that plasma concentrations of GSH in the premature infants were substantially, indeed often dramatically, lower than we have observed in adult humans (6 to 10 μM). Finally, we found that in patients with both umbilical arterial and umbilical venous catheters arterial GSSG concentrations were consistently higher than venous concentrations; conversely, arterial GSH concentrations were lower than venous concentrations. The elevated GSSG concentrations we observed in these infants indicate marked oxidant stress responses in prematurely born infants, even in those infants exposed only to room air. The positive arteriovenous gradients of GSSG concentrations across the lungs of these infants suggest that at least some of the increased plasma GSSG originates in the lung. The low plasma GSH concentrations we observed in these same infants suggest deficiencies in an antioxidant that has been shown in numerous animal studies to be critical for prevention of hyperoxia-induced lung injury. Finally, the negative arteriovenous gradients of GSH concentrations across the lung provide the first evidence in humans for pulmonary uptake of GSH.

Hypoxic damage generates reactive oxygen species in isolated perfused rat liver

The aim of the present study was to investigate the possible role of reactive oxygen species in the pathogenesis of hypoxic damage in isolated perfused rat liver. One hour of hypoxia caused severe cell damage (lactate dehydrogenase release of greater than 12,000 mU/min/g liver wt) and total irreversible cholestasis which was accompanied by a loss of cellular ATP and a marked decrease in lactate efflux. Tissue glutathione disulfide (GSSG) content and GSSG efflux as a measure of hepatic reactive oxygen formation was less than 1% of total glutathione before and during hypoxia. Upon reoxygenation, however, hepatic GSSG content increased sharply to about twice the control values and GSSG efflux increased several-fold to around 3-4 nmol GSH-equivalents/min/g. The release of lactate dehydrogenase decreased upon reoxygenation and tissue ATP content recovered partially. When livers were reoxygenated at an earlier time interval than 1 hr of hypoxia, i.e., before the onset of damage, no enhanced GSSG formation was observed. The results demonstrate that hypoxic damage is a prerequisite to reactive oxygen formation during the subsequent reoxygenation period. Thus, reactive oxygen species appear unlikely to play a crucial role in the pathogenesis of hypoxic liver damage in the hemoglobin-free, isolated perfused liver model.

High-performance liquid chromatography and gas chromatography-mass spectrometry determination of specific lipid peroxidation products in vivo

Peroxidation of membrane lipids has been implicated in the toxicity of reactive oxygen intermediates and of several hepatotoxins, but the specific products of this peroxidation in vivo have not been chemically identified. A method for the isolation, identification, and quantitation of specific lipid hydroperoxy and hydroxy acids formed in vivo has been developed. Hydroxylated derivatives of linoleic, arachidonic, and docosahexaenoic acids formed in mouse liver phosphatidylcholines following carbon tetrachloride administration were isolated by high-pressure liquid chromatography and identified as the trimethylsilyl ether methyl ester derivatives by gas chromatography-mass spectrometry. This methodology should be important for the investigation of the role of lipid peroxidation in a variety of normal physiologic and pathologic processes.

Quantitation of lipid peroxidation products by gas chromatography-mass spectrometry☆

A method for the quantitation of lipid peroxidation products in total hepatic lipid has been developed. Lipid extracts are reduced and subsequently transmethylated with sodium methoxide. The hydroxy fatty acid methyl esters are isolated by silicic acid chromatography and derivatized to their trimethylsilyl ethers prior to analysis by gas chromatography-mass spectrometry. Three isomers, 11-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE), are quantitated using selected ion monitoring techniques relative to the internal standard, methyl 15-hydroxyarachidate. In mice treated with carbon tetrachloride (2 ml/kg), the HETE levels in total hepatic lipid were 20-fold greater than those found in control animals. HETE levels were also elevated (5- to 10-fold) in hepatic lipid from rats treated with the same dose of carbon tetrachloride. Studies on subcellular fractions with this methodology show that these lipid peroxidation products are 5- to 6-fold higher in hepatic plasma membrane vesicles isolated from rats treated with carbon tetrachloride when compared with those isolated from control animals.

Alkylation of the liver plasma membrane and inhibition of the Ca2+ ATPase by acetaminophen

Acetaminophen is activated metabolically to yield reactive species that bind covalently to liver cell macromolecules. The extent of covalent binding correlates with the occurrence and severity of hepatic necrosis. We reported previously [J. O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)] that active Ca2+ accumulation of isolated liver plasma membranes is decreased 60-75% after a hepatotoxic dose of acetaminophen in vivo. We now report that the protein of isolated liver plasma membranes was substantially labeled with drug metabolites after administration of [3H]acetaminophen. There was no increase in passive membrane permeability that might cause diminished Ca2+ accumulation. Intravesicular volume and relative purity of the vesicle preparations after acetaminophen were not different from controls. However, (Ca2+,Mg2+)-ATPase, a possible biochemical expression of the Ca2+ pump, was decreased 31% (P less than 0.025) after acetaminophen treatment. ATPase activity in both control and treated groups was enhanced by isolating membranes in the presence of 5 mM reduced glutathione (GSH), but the effects of drug treatment were not reversed. A similar effect of GSH on Ca2+ accumulation was observed previously [J. O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)]. These data are consistent with a hypothesis wherein alkylation of membrane proteins by reactive acetaminophen metabolites is a factor in the onset of hepatic necrosis after acetaminophen. They are not consistent with an oxidative stress hypothesis where thiol S-thiolation of membrane components is postulated to produce altered membrane permeability or thiol-reversible alterations in membrane protein structure and enzymatic function.

Isolation, Characterization, and Functional Assessment of Oxidatively Modified Subfractions of Circulating Low-Density Lipoproteins

Objective—Current evidence suggests that oxidatively modified human plasma low-density lipoproteins (ox-LDLs) are proatherogenic and cytotoxic to endothelial and vascular smooth muscle cells. The present study describes a method using ion-exchange chromatography that is capable of large-scale subfractionation of LDL for adequate analyses of composition or bioactivities. Methods and Results—LDLs from normolipidemic (N-LDL) and homozygous familial hypercholesterolemic (FH-LDL) subjects were separated into 5 subfractions (L1 through L5) by high-capacity ion-exchange chromatography. The most strongly retained fraction from FH subjects, FH-L5, suppressed DNA synthesis in cultured bovine aortic endothelial cells and stimulated mononuclear cell adhesion to cultured endothelial cells under flow conditions in vitro. L5, which represented 1.1±0.2% and 3.7±1.7% of the LDL from N-LDL and FH-LDL, respectively, was more triglyceride-rich (17% versus 5%) and cholesteryl ester-poor (23% versus 33%) than were L1 through L4. Electrophoretic mobilities on agarose gels increased from L1 to L5. According to SDS-PAGE, apolipoprotein B-100 in N-LDL fractions L1 through L5 appeared as a single ≈500-kDa band. In contrast, the fractions isolated from FH-LDL showed substantial fragmentation of the apolipoprotein B-100, including bands between 200 and 116 kDa. Competitive ELISA analyses using a malondialdehyde-specific monoclonal antibody against Cu2+ ox-LDL suggest that FH-L5 is malondialdehyde-modified. Conclusions—Relative to N-LDL, FH-LDL contains higher concentrations of a fraction, L5, that exhibits distinctive physicochemical properties and biological activities that may contribute to initiation and progression of atherogenesis in vivo.

Methods for determination of lipid peroxidation in biological samples

Interest in the pathological consequences of lipid peroxidation has led to the development of a number of analytical approaches to the quantitation of products. However, the various analytical methodologies employed often do not measure the same chemical classes of products, and apparent discrepencies have been observed, particularly in studies of lipid peroxidation in biological systems. This review provides a brief discussion of some of the strengths and weakness of methods currently used for the determination of lipid peroxidation in biological tissues.

Acetaminophen hepatotoxicity invivo is not accompanied by oxidant stress

Hepatotoxic doses of acetaminophen in Fischer 344 rats did not increase biliary efflux of oxidized glutathione. Pretreatment of the animals with bis(2-chloroethyl)-N-nitrosourea inhibited hepatic glutathione reductase by 73 percent but did not potentiate the hepatotoxicity of acetaminophen and did not produce an increase in biliary efflux of oxidized glutathione in response to acetaminophen. Hepatic protein thiol content was not depleted by acetaminophen. A proposed role for oxidant stress mechanisms mediated either by reactive oxygen species or by the direct oxidant action of a reactive metabolite in acetaminophen-induced hepatotoxicity is unsubstantiated and unlikely.

OXIDATIVE MODIFICATIONS OF APOB-100 BY EXPOSURE OF LOW DENSITY LIPOPROTEINS TO HOCL IN VITRO

Although the products of oxidation of the lipid components of LDL have been studied extensively, much less is known about the specific products of oxidative modification of the apoprotein. We reacted native LDL and LDL that had been treated with HOCl with 2,4-dinitrophenylhydrazine (DNPH), delipidated and trypsinized the protein, and analyzed the products by HPLC. Although tryptic digests of native LDL and LDL oxidized by limited quantities of HOCl showed similar patterns by HPLC with detection at 220 nm, oxidized LDL showed several discrete peaks at 365 nm, which is characteristic of hydrazones formed with aldehydes and ketones, commonly termed protein carbonyls. Native LDl showed no peaks in the chromatograms at 365 nm. Peptides absorbing at 365 nm were isolated by HPLC and characterized. In most cases, the probable sites of modification on the peptides could be implied by failure of an anticipated amino acid to appear in the expected sequence. Of the 14 peptides isolated and characterized to date, eight peptides contained Cys residues. In other peptides, Lys, Trp, and Met were identified as amino acid residues apparently modified by HOCl treatment of LDL. Thirteen of the peptides identified are from trypsin-releasable peptides located on the surface of unoxidized native LDL. Our studies suggest a selective process of modification of apoB-100 by HOCl and the approaches used in the present studies should be useful for the characterization of the mechanisms of oxidation of this and other proteins.

Induction and decline of hepatic cytochromes P4501A1 and 1A2 in rats exposed to hyperoxia are not paralleled by changes in glutathione S-transferase-α

We investigated the effects of hyperoxia on the activities of hepatic ethoxyresorufin O-deethylase (EROD) (CYP1A1), methoxyresorufin O-demethylase (MROD) (CYP1A2), and glutathione transferase-alpha (GST-alpha), and the status of protein thiols (PSH) in male Sprague-Dawley rats. Twenty-four h of hyperoxia more than doubled EROD and MROD activities, which were increased 7.6- and 3.3-fold, respectively, after 48 h of hyperoxia. The increases in EROD and MROD activities were paralleled by enhanced CYP1A1/1A2 apoproteins contents, as detected by Western analysis. At 60 h of hyperoxia, by which time hyperoxic Sprague-Dawley rats display marked respiratory distress, pulmonary edema, and other markers of pulmonary dysfunction, the activities and levels of hepatic CYP1A1 and 1A2 had declined dramatically and returned to levels observed in air-breathing control animals. Hepatic activities of GST-alpha, as well as PSH status, were not altered significantly in the hyperoxic animals at any time point. The marked induction and subsequent decline of hepatic CYP1A1/1A2 activities in rats exposed to hyperoxia suggest that these enzymes may contribute to the mechanisms of injury and/or to adaptive responses to hyperoxic exposures in vivo.