|
Among healthy individuals under oxidative stress
conditions GSH interacts with the oxygen radicals formed and significantly
decreases the harmful effects of such reactive species. As GSH homeostasis
decreases from the normal level the lung is susceptible to serious
oxidative damage. Proteins found in the lung lining are also vulnerable
to these destructive oxidant radicals. In order to locate modified
proteins of the lung lining protein size and lipid breakdown products
(malonyldialdehyde; MDA) were analyzed for surfactant protein A
(SP-A) which is responsible for fighting lung infection. Surfactant
is a pulmonary phospholipid substance important in controlling the
surface tension of air-liquid emulsion that is present in the lungs.
It is our hypothesis that chronic alcohol abusers are at risk for
protein radical formation with SP-A. This surfactant protein is
also important in stimulating alveolar macrophage to phagocytize
and kill infectious particles. Therefore altered SP-A would impair
both the binding of infectious particles as well as their uptake
and clearance by alveolar macrophages. We anticipated that chronic
oxidative stress resulting from alcohol abuse would cause the oxidation
of SP-A.
Alterations in the pulmonary glutathione (GSH) homeostasis
due to chronic alcohol abuse causes the development of the acute
respiratory distress syndrome (ARDS) and therefore oxidative stress.
Under ARDS conditions lipids and proteins are transformed to oxidative
radicals which are deleterious because they bind to proteins at
cysteine disulfide bridges and render the protein inactive.
Human lung wash (lavage) samples were collected from
patients with and without a history of alcohol abuse. The lavage
of several different subjects were determined by western blot and
dot blot. The proteins were probed with rabbit anti-SP-a antibody
(1:5000) to determine the size of the SP-a present as well as goat
anti-MDA antibody (1:5000) to determine the amount of lipid breakdown
product adducted to SP-A. The sizes of the protein and the levels
of MDA/SP-a adducts from the control patients without a history
of alcohol abuse were compared to the levels of the patients that
abused alcohol.
Development of the western blot [Figure 1] showed
that there was no difference between the size of the SP-A between
the controls and alcoholics. If the protein had been modified it
would have shifted from the polymeric form to a smaller aggregate
as the tertiary structure is compromised upon oxidation. In addition
the dot blot [Figure 2] confirmed that the levels of lipid breakdown
products (MDA) from those with or without a history of alcohol abuse
were similar. Had the SP-A from alcoholics been oxidized a greater
intensity of MDA would have been expressed in the alcoholic samples
since lipid breakdown products indicate oxidation.
The results of the experiments refuted the hypothesis that the
chronic oxidative stress caused by alcohol abuse resulted in surfactant
protein A modification. In fact alcoholic SP-A does not undergo
any more serious modifications by oxidative radicals than the surfactant
protein of non-alcohol abusers. Therefore in the alveolar space
SP-A is not significantly modified under oxidative stress conditions.
Future directions include researching the possible physiological
modifications of the other three surfactant proteins (B C and D)
under oxidative stress conditions.
This material is based upon the work supported by the Howard Hughes
Medical Institute under Grant No. 52003727. The help throughout
the summer from the Summer Undergraduate Research Experience program
at Emory (SURE) is greatly appreciated.
|
