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Fasted mice exposed to 100% oxygen have more lung damage and die sooner than do fed mice. The mechanism responsible for this phenomenon has not been identified. We performed the following experiments to test the hypothesis that reduced glutathione content in lung tissue of fasted mice contributes to the increased susceptibility to hyperoxic lung damage. First, air-exposed mice were fasted for as long as 3 days. They had little change in lung levels of superoxide dismutase (SOD) or catalase, but they had a 41% decrease in glutathione by Day 3 (p less than 0.001). Second, fed mice and fasted mice were exposed to 100% oxygen for as long as 4 days. Both groups had nearly identical values of lung SOD and catalase, but the fasted mice had lower levels of glutathione (p less than 0.001). Third, fed mice received the glutathione synthesis inhibitor buthionine sulfoximine (BSO; 20 mM) in their drinking water for 2 wk and were then exposed to either air or 100% oxygen. Air-exposed mice receiving BSO for 14 days had no change in lung SOD content, a 43% increase in catalase (p less than 0.001), and a 41% decrease in glutathione (p less than 0.01). Oxygen-exposed, BSO-treated mice had no change in SOD and an increase in catalase, but lower glutathione levels, more deaths, and increased lung damage on Day 3 (BAL protein: 1.72 +/- 0.21 versus 0.94 +/- 0.08 mg/ml; p less than 0.01) than did diluent-treated mice. Fourth, fasted mice were given liposomes containing glutathione intratracheally.(ABSTRACT TRUNCATED AT 250 WORDS)

To gain insight into how glutathione given directly into the lung protects fasted mice against hyperoxic lung damage and to provide a framework for developing treatment strategies in patients, we determined the lung distribution and retention of intratracheally administered glutathione (GSH) and its fate after leaving the lung. Mice received an intratracheal injection of [3H]GSH with or without cold GSH and with or without liposomes. The distribution of the 3H label was equal in both lungs, but the left lung had a higher concentration because of its smaller size. The 3H label was cleared rapidly from the lung: only 1% remained at 24 h, and more than 50% of the label at that time was no longer attached to the GSH. Administration of GSH with liposomes increased the retention of GSH by 20 to 50%, but the amount remaining at 24 h was still only 1%. The increase associated with the liposomes was due to enhanced retention of the GSH encapsulated in the liposomes, not the much larger amount present free in the GSH-liposome mixture. Fasting and exposure to 100% oxygen had little effect on GSH retention. Some of the 3H label leaving the lung was excreted by the kidneys, a small amount was retained in the liver, and a large amount accumulated in the blood. Of the amount in the blood, about 60% was in red blood cells (RBC) and the rest in plasma. Much of the 3H label in RBC and lung at 24 h was no longer attached to the GSH, whereas most in the plasma and liver was.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspirin acetylates a variety of sites on both the a and ft chains of hemoglobin S. Nevertheless, the vast majority of acetyl groups become attached to three loci:flLys 59, ,6Lys 144, and aLys 90. These observations reveal some molecular details of this transacylation reaction and suggest interesting possibilities for its extension to other acylsalicylates with a variety of different structures.We have reported previously (1) that incubation of sickle cell hemoglobin (HbS) (or of HbA) with aspirin leads to incorporation of acetyl groups into the protein. This observation has been confirmed since then by others (2). On the other hand, it has been reported that this acetylation does not change the oxygen affinity of hemoglobin (2), contrary to our original impression (1). If the NH2-terminal amino groups were acetylated, one would expect the oxygen affinity to be modified, since carbamylation of the Val-1 residues has such an effect (3). In any event, to provide more details on the nature of the reaction of aspirin with HbS and its potential to interfere with sickling, we have carried out enzymatic hydrolyses of the labeled protein and determined the sites of residence of the major portion of the covalently linked acetyl groups. MATERIALS AND METHODSIncorporation experiments were carried out at 370 at pH 7.2 in 50 mM buffer made from Bis-Tris [N,N-bis(2-hydroxyethyl)iminotris(hydroxymethyl)-methane]. Aspirin was near 20 mM, and HbS, stripped of 2,3-diphosphoglycerate (4), was adjusted to 2% final concentration. Radioactively labeled aspirin ("4C in the carboxyl group of acetyl, 6.60 Ci/mol and warranted isotopic purity of 98%) was obtained from Mallinckrodt Chemical Works. The extent of incorporation of ['4C]acetyl groups was measured by liquid scintillation counting, following the procedure described (1).To locate the position of the labeled acetyl groups on hemoglobin, we used standard procedures for peptide separation and analysis. Chromatographic fractionation of hemoglobins was performed essentially as described by Dozy et al. (5). Hemoglobin samples (40-80 mg) were flushed with carbon monoxide, adjusted to pH 8.5 with 50 mM Tris-HCl buffer (pH 10), and applied to a 50 X 1.2-cm column of DEAESephadex. The column was equilibrated with a buffer of Tris HCl (pH 8.3), containing KCN (100 mg/liter). Chromatography was performed with a linear Trist HCl buffer gradient decreasing from pH 8.3 to pH 4.5. The effluent fractions comprising each of the hemoglobin peaks were pooled and concentrated by centrifugation overnight at 100,000 X g.Globin was prepared from the hemoglobin fractions by precipitation in acetone-HCl at -20°(6). Globin chain fractionation was performed by column chromatography on carboxymethyl-cellulose at pH 6.7 in 8 M urea as described by Clegg et al. (7). The protein-containing effluent fractions were pooled and treated with ethyleneimine (7). Urea and other low-molecular-weight substances were removed from the globin solutions by gel filtration through a column of Biogel P-2 equilibrated wi...

An electrophoretically slow-moving hemoglobin variant was identified in three members of a family originating from Southern Mexico. The variant, Hb Lincoln Park, made up approximately 14% of the total hemoglobin and appeared to gave normal stability and functional properties.None of the individuals in whom the abnormal hemoglobin was present was anemic, but each had a mildly elevated reticulocyte count. Structural data suggest that the non-a chain of Hb Lincoln Park represents a #5 gene-fusion product, with normal chain structure of the amino-terminal portion of the chain and sequences subsequently, the crossover point occurring between amino acid residues 22 and 50. An 7). Oxygen equilibrium curves were determined with a blood gas analyzer, model 217, from Instrumentation Laboratories, Inc. The procedures were as described (8). For determinations of 2,3-diphosphoglycerate, protein-free filtrates were prepared from trichloroacetic acid-treated samples of whole blood. The spectrophotometric assay procedure was as described by Keitt (9). Isolation of the Variant Hemoglobin and Globin Chain. DEAE-Sephadex column chromatography of carbon monoxide-saturated hemoglobins was performed according to Dozy et al. (10). Effluent fractions corresponding to the variant hemoglobin were pooled and the hemoglobin was concentrated by vacuum dialysis. Globin was prepared by precipitation in acetone/HCI at -20°, and the globin chains were separated by CM-cellulose chromatography (11). Globin representing the non-a chain was aminoethylated (11) and subsequently desalted by gel filtration through a column of Bio-Gel P-2 equilibrated with 0.5 M formic acid. The protein was recovered by lyophilization.Peptide Isolation and Purification. The aminoethylated globin was digested with trypsin for 2 hr at 370 in NH4HCO3 buffer. The tryptic peptides were separated by PA-35 column chromatography with a linear pyridine/acetic acid gradient (12). Peptides requiring repurification were subjected to chromatography on a column of Dowex 50-X2 with the same buffer system. Amino Acid Analysis. Purified samples of globin and peptides for amino acid analysis were hydrolyzed for 24 hr, or in some cases 72 hr, in 6 M HCI under reduced pressure at 1100. The analyses were performed with a Beckman-Spinco model 121M amino acid analyzer equipped with a system AA integrator. Sequence analysis of the abnormal peptide was by a subtractive Edman procedure based on the method of Gray (13) with minor modifications. RESULTSCase Report. The index case, a 3-year-old child, was referred for evaluation when he was found to have an electrophoretically slow-moving abnormal hemoglobin by a municipal hemoglobinopathy survey. The child had been in good health and had