The overall degree of protein oxidation in BALF was significantly higher in CF patients than in healthy subjects (Fig 1). CF patients with pathologic FEV1 (< 80% of predicted) had considerably higher levels of protein carbonyls (median, 3,033 pmol/mL BALF; 15.6 pmol/^g protein) compared to either CF patients with normal pulmonary function values (FEV1 > 80% of predicted; 612 pmol/mL; 6.9 pmol/ ^g; p < 0.01) or healthy control subjects (0 pmol/ mL, p < 0.001) [Fig 1].
There was an inverse correlation between pulmonary function and the level of protein carbonyls in the BALF of CF patients (Fig 2). Similarly, CF patients with a high degree of neutrophilic inflammation (> 10% of PMNs in BALF) had exceedingly higher values of protein carbonyls (2046 pmol/mL; 11.5 pmol/^g) than those with a low neutrophil count (< 10%; 53.8 pmol/mL; 0.5 pmol/^g; p < 0.001) and the control group (p < 0.001) [Fig 1]. As expected, protein carbonyls and total cell count in BALF (n = 52; rs = 0.54; p < 0.0001), absolute neutrophil count (n = 46; rs = 0.62; p < 0.0001), and relative neutrophil count (n = 47; rs = 0.6; p < 0.0001; Fig 3) were positively correlated within the group of CF patients.
The distribution of overall protein oxidation was evaluated in order to investigate if certain proteins are selectively oxidized while others are spared. This was performed by means of two-dimensional electrophoresis techniques with post-isoelectric focusing DNPH derivatization and Western blotting.
Both the number and volume (optical density times area) of oxidized proteins increased with increasing overall degree of oxidation of a sample (see representative gels in Fig 4). However, no clear hierarchic sequence of preferential oxidation of certain protein spots could be identified with the gels from CF patients with different degrees of overall oxidation. More info
Figure 1. Protein carbonyls in BAL. The group of CF patients (n = 55) had a significantly higher level of protein carbonyls in BALF in comparison to the control group of healthy subjects (n = 11) [Mann-Whitney U test; upper panel]. When the CF patients were subgrouped according to pulmonary function (FEV1) values, there was a significant difference in the content of protein carbonyls within the group of CF patients (Kruskall-Wallis test with Dunn post hoc test; middle panel). Similarly, when the CF patients were classified into subgroups with high (> 10%, n = 30) and low (< 10%, n = 16) content of neutrophil granulocytes (PMNs) in their BAL, the protein carbonyl values were also different (lower panel).
Figure 2. Lung function parameters were inversely correlated to the level of protein carbonyls in BALF of patients with CF. MEF25 = maximum expiratory flow at 25% of vital capacity; MEF25-75 = maximum expiratory flow between 25% and 75% of vital capacity.
Figure 3. Protein carbonyls in the group of the CF patients were positively correlated with the fraction of neutrophils in their BALF.
Figure 4. Pattern of BALF protein oxidation in CF. Shown are Western blots of two-dimensional electrophoresis gels of BAL samples from CF patients with low (< 100 pmol/mL BALF protein carbonyls, left panel) and high (3,033 pmol/mL BALF protein carbonyls, right panel) grades of protein oxidation (anti-DNPH immunostaining; representative of six gels are shown).