There was a significant increase in the number of cells per milliliter of bronchoalveolar lavage after sham operation (p < .05) and liver I/R (p < .01) compared with normal controls. The cell differential resulted in a significant increase in the percentage of bronchoalveolar lavage neutrophils after I/R (48% ± 26%) compared with sham-operated controls (5% ± 3%) (p < .01) ().
The TNF-α concentration in plasma of I/R-treated rats was 219 ± 180 pg/mL (n = 6) (), whereas the cytokine had an average content of 7 ± 15 pg/mL in the plasma of normal controls (n = 5). TNF-α was not detected in plasma samples from sham-operated rats. The IL-8 analog CINC-1 was significantly elevated in the plasma of I/R-treated animals (1537 ± 279 pg/mL; n = 6) () compared with both normal controls (204 ± 128 pg/mL; p < .0001) and sham-operated rats (930 ± 140 pg/mL; p < .001). The difference in plasma CINC-1 content between normal controls and sham-operated rats was statistically significant as well (p < .0001).
Figure 1 A, tumor necrosis factor (TNF)-α concentration in the plasma. The TNF-α concentration in plasma of ischemia-reperfusion (I/R)-treated rats was significantly elevated at 219 ± 180 pg/mL (n = 6), whereas the cytokine had an average (more ...)
Mass Spectrometry Measurements
Data were obtained for 1,513 proteins. Ninety-four of these proteins met the threshold criteria for quantitation and statistical comparison in the I/R-treated group and 97 in the sham-operated group. On average, the quantified proteins were identified on the base of 29 ± 6 peptides. Testing for multiple comparisons by FDR analysis resulted in an FDR threshold (α) value >.002 for the I/R-treated group and .001 for the sham-operated group. In the statistical comparison between the nonoperated control samples to evaluate variability for the proteins that met the thresholds for quantitation in the I/R group, five of 95 Student’s t-tests were statistically significant (5.2%), whereas four of 80 (5%) tests were statistically significant for the proteins that were quantified in the sham-operated animals.
Catalase ( and ) was present in a 34% increased concentration in the rats after I/R (p < .0001), whereas there was no significant difference in its content between sham-operated rats and normal controls. The difference in its content between the I/R and sham-operated groups was significant as well (p < .0001). There was a 52% increase in the cellular content of myeloperoxidase in rats after I/R (p < .0001). The enzyme content was elevated to a lesser degree, but the increase was statistically significant (p < .0001), in animals after sham operation. The difference between the I/R and the sham groups was also significant (p < .0001). Only one isoform of superoxide dismutase, [Cu-Zn], was quantified. Compared with controls, the content of superoxide dismutase [Cu-Zn] was not altered in the I/R or the sham-operated groups (p not significant), although there was a significant difference in the direct comparison of the two groups (p < .0005).
Proteins of the oxidant system
Figure 2 Proteins of the oxidant system. The proteins catalase, myeloperoxidase, and superoxide dismutase were significantly elevated in alveolar epithelial type II cells from rats after liver ischemia-reperfusion (I/R) but not after sham operation. The bars represent (more ...)
Adenosine Phosphate Generating Enzymes
All quantified enzymes of the adenosine triphosphate (ATP)-generating pathways followed the same pattern. In rats after both liver I/R and sham operation, a similar and statistically significantly elevated content in the cellular content of adenosine diphosphate/ATP translocase 2 of 37% (p = .0001) and 28% (p < .0001) ( and ) was found. An increased content in the β chain of the mitochondrial ATP synthase was observed in rats after liver I/R (31%, p < .0001) and sham operation (16%, p < .0001). The α chain of the mitochondrial ATP synthase was increased by 39% in the liver I/R group (p < .001) and by 24% after sham operation (p < .0001).
Figure 3 Enzymes of the adenosine phosphate system. The three enzymes proteins adenosine triphosphate (ATP) synthase α chain, ATP synthase β chain, and adenosine diphosphate (ADP)/ATP translocase 2 were significantly elevated in rats after both (more ...)
Enzymes of the adenosine phosphate system
The protein content of the quantified cellular enzymes followed two distinct patterns: Nine of these enzymes were statistically significantly up-regulated in rats after liver I/R, whereas the cellular content in animals after sham operation was not significantly changed ( and ). This group consisted of aldehyde dehydrogenase (43% increase with I/R, p < .0001), aspartate aminotransferase (42% increase, p < .0001), carboxylesterase 3 (42% increase, p < .0001), fatty acid synthase (39% increase, p < .0001), L-lactate dehydrogenase (49% increase, p < .0001), mitochondrial malate dehydrogenase (43% increase, p < .0001), and three isoforms of the protein disulfide isomerase (A3, 64% increase, p < .0001; A6, 43% increase, p < .0005; and precursor, 32% increase, p < .0001). The cellular content of the remaining enzymes did not change statistically significantly after any of the interventions.
Figure 4 Intracellular metabolic enzymes. There were significantly increased cellular contents in glucose regulating proteins (GRP) 75 and 78, malate dehydrogenase, L-lactate dehydrogenase (A-chain), and fatty acid synthase in rats after liver ischemia-reperfusion. (more ...)
Quantitative information for several cell regulatory proteins () was obtained. The cellular content changes that were observed in these regulatory proteins in rats with and without liver injury can be subdivided into three different patterns. The contents of a first group were statistically significantly increased in the group after liver I/R but not in the group after sham operation. These were 78-kDA steroidogenesis activator polypeptide (46% increase, p < .0005), calregulin (35% increase, p < .0001), elongation factor 1-α1 (25% increase, p < .001), mitochondrial 10-kDA heat shock protein (Hsp) (55% increase, p < .003), Hsp 60 (54% increase, p < .002), annexin A5 (36% increase, p < .0001), protein kinase C inhibitor protein 1 (50% increase, p < .001), and tumor rejection antigen gp 96 (28 increase, p < .0001). The cellular contents in a second group of proteins were significantly increased in both the liver I/R and the sham operation groups. Members of this group were members of the annexin family (A1, 46% increase in animals after I/R, p < .0001, and 23% increase in rats after sham operation, p < .001; A6, I/R +35%, p < .0001 and sham +16%, p < .0001). The cellular content of the remaining proteins was not statistically significantly altered in rats after any intervention, although some of the remaining proteins seemed to have a noticeable but not statistically significant trend toward an increased cellular content in rats after liver I/R. These proteins included annexin A2 (I/R +33%, p < .02, and sham +31%, p = .001) as well as calcyclin, heat shock cognate 71-kDa protein, Hsp 90 β, lymphocyte cytosolic protein 1, prohibitin, and ubiquitin.
Measured differences in the cellular content in structural proteins () could be subdivided into two different patterns. The first group of proteins was significantly increased in the group after liver I/R but not in the group after sham operation. These proteins were β-actin (26% increase, p < .0005), cytokeratin 8 (52% increase, p < .0001), lamin A (22% increase, p < .0001), lamin B1 (50% increase, p < .0001), moesin (20% increase, p < .001), α-spectrin (44% increase, p < .0005), β spectrin (45% increase, p < .0001), and vimentin (44% increase, p < .0001). The cellular content of other proteins was significantly increased in the animals that had undergone either liver I/R or sham operation. Members of this group were myosins (M-14: I/R +47%, p < .0001; sham +22%, p < .0001; M-9: I/R 347%, p < .0001, sham +20%, p < .0001) and plectin-1 (I/R +43%, p < .0001; sham +30%, p < .0001).