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Background The consumption of alcohol
can cause a multitude of health problems among those individuals
who participate in the abuse of this substance. Among the many health
problems is acute respiratory distress syndrome, or ARDS. ARDS can
lead to severe respiratory problems and has been noted to lessen
the survival rate of trauma patients that were affected with this
syndrome. In order to measure the severity of ARDS two methods have
been proposed. These methods involve an assessment of hydrogen peroxide,
H2O2, and albumin in the lung fluid.
Materials and Methods After informed
consent, a flexible fiber optic bronchoscope was wedged in the right
middle lobe of all subjects by a trained pulmonologist. Sterile
saline (150 mL) was injected and immediately retrieved (termed lavage).
In order to find an alternative method of determining ARDS in alcoholic
patients, trauma patients, and smokers we measured the amount of
H2O2 and albumin in the bronchoalveolar lavage (BAL). For H2O2,
using a PeroXOquant peroxide kit and a spectrophotometer, we determined
the amount of H2O2 in the human BAL. In order to establish a standard
curve several serial dilutions were made from the stock solution
of 43 mM H2O2. Based on these serial dilutions, we interpolated
the H2O2 concentration of the BAL samples. For albumin, standard
ELISA protocol and a spectrophotometer were used to determine the
concentration of albumin in the human BAL. For the standard curve,
several serial dilutions were made from the stock solution of 200
ng/uL of human albumin. The lavage procedure dilutes the epithelial
lining fluid. To control for dilution, H2O2 and albumin measurements
were normalized to the BAL concentration of the protein immunoglobulin
A (IgA). This protein was chosen because IgA is a secretory protein
that is unchanged during injury. An ELISA was used to determine
the IgA concentrations in the BAL. This was accomplished by comparing
the levels of H2O2 (nmol/uL) and IgA (ng/uL) in a ratio that yielded
a concentration of nanomoles of H2O2 to nanograms of IgA. The concentration
levels of albumin in ng/uL were also compared to IgA in ng/uL in
a ratio that yielded a concentration in ng of albumin per ng of
IgA. After the normalization was completed the BAL was then termed
epithelial lining fluid (ELF).
Results and Discussion When the lavage
samples were tested there were several trends that were evident.
This became clearer when the normalization results were entered
into the Sigma Plot program. The otherwise healthy alcoholics had
more H2O2/IgA than the smokers and the controls (with a range of
.39-3.3 nmol/uL). Some otherwise healthy alcoholics were lavaged
a second time after one week of abstinence from alcohol consumption.
There was trend towards decreased H2O2 (n=10; p=0.161) after abstinence
but additional subjects will need to be assessed to achieve statistical
significance. In the lavage from alcoholic ARDS patients, H2O2 was
greater than ARDS patients without a history of alcohol abuse (p=0.179)
but additional subjects are needed to achieve statistical significance.
This suggested that pulmonary oxidative stress was greater in subjects
with a history of alcohol abuse. To assess pulmonary injury, the
leak of the vascular protein albumin into the air space was assessed.
In otherwise healthy alcoholics, the lavage albumin was greater
than that present in controls and smokers (p < or = 0.05). This
suggested that when controlled for smoking, alcohol abuse resulted
in lung injury even in otherwise healthy subjects. Abstinence from
alcohol ingestion trended towards decreased albumin leak but additional
subjects are needed. When the H2O2 concentration in the ARDS was
plotted against the albumin leak, there was a positive correlation
and suggested that H2O2 was a good marker for lung injury. Since
H2O2 is exhaled, the H2O2 content in a breath condensate may serve
as a simple non-invasive marker for lung injury.
Key Words Acute Respiratory Distress
Syndrome, Bronchioalveolar Lavage, Alcoholism, Smoking, Hydrogen
Peroxide, Albumin
Alcohol abuse causes a multitude of health problems.
When superimposed on trauma, aspiration or sepsis, alcohol abuse
increases the risk of morbidity and mortality from lung failure
termed acute respiratory distress syndrome, or ARDS. Alcohol abuse
decreases in the availability of the antioxidant glutathione and
results in oxidative stress. We propose that the greater the oxidative
stress, the greater the risk of pulmonary injury and ARDS. If this
hypothesis is true, then the oxidant hydrogen peroxide (H2O2) in
lung fluid may be a measure of the risk or the severity of ARDS.
Furthermore, preliminary data has demonstrated that the chronic
oxidative stress injury to the lungs results in an increase in protein
levels in the lung fluid collected from healthy alcoholics. This
suggests that chronic alcohol abuse causes sub-clinical injury to
the lungs and results in vascular leak into the airspace. A flexible
fiberoptic bronchoscope was wedged in the right middle lobe of control,
alcoholic and ARDS volunteers. A lung lavage was performed by injecting
150 ml of sterile saline into the lobe and aspirated into a suction
trap. A second lavage was performed on some alcoholics after one
week of abstinence. We measured the H2O2 in the lavage fluid. Using
ELISA techniques, we quantitated the plasma protein albumin in the
lavage fluid.
Hydrogen Peroxide Assay:
- PeroXOquant Quantitative Peroxide Assay Kit was used to measure
the amount of H2O2 in the lavage samples from alcoholics, smokers,
nonsmokers and ARDS patients.
- Standards were prepared by diluting the H2O2 stock solution
of 43.6 mM.
- The stock was diluted to 1mM by adding 23.7 uL of the 43.6 mM
H2O2 to 977 uL coating buffer. Then this was serially diluted
to 10 uM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.3 nM, 3.2 nM, 1.6 nM,
and 0.8 nM.
- Using the 96 well high binding plate, 25 uL of each standard
and lavage samples were added to a well in duplicate.
- Reagent: 1 volume of reagent A was mixed with 100 volumes of
reagent B.
- Reagent was added to the sample or standard at a ratio of 10
volumes of reagent to 1 volume of samples.
- Samples and standards reacted with this reagent for 15 minutes
- Absorbance of the samples and reagent mix was read at 590 nm
using the Spectra-Fluor spectrophotometer.
Albumin ELISA:
- An ELISA was performed on the lavage samples from the alcoholics,
smokers, nonsmokers, and ARDS subjects.
- The Albumin stock (200 ng/uL) was diluted to 4 ng/L and serially
diluted to 2, 1, .5, 0.25, 0.125, and 0.63 ng/uL.
- 100 uL of the standards in duplicate were added to a well
- 10 uL of sample and 90 uL of coating buffer were added to a
well in duplicate.
- Samples/standards were incubated for 1 hour at room temperature
on the shaker (100-150 rpm).
- Samples/standards were washed three times with wash buffer.
- 200 uL of blocking buffer were added to each well and incubated
for 30 minuets at room temperature on the shaker.
- After the plate was emptied, 100 uL of primary antibody was
added and the samples/standards were incubated at room temperature
for 2 hours on the shaker.
- Samples/standards were washed three times with the wash buffer.
- After the secondary antibody was added, the samples/ standards
were incubated for 30 minutes at room temperature on the shaker.
- Wells were then washed three times with the wash buffer.
- Color developing substrate was added and incubated for 30 minutes.
- Stop reagent was added and the absorbance was read at 450nm.
Protein Assay:
- Standards of human albumin were prepared to concentrations of
1000 ug/mL, 750 ug/mL, 500 ug/mL, 375 ug/mL, 250 ug/mL, 125 ug/mL,
62.5 ug/mL, and 12.5 ug/mL.
- 10 uL of deionized water was added to each well containing 10
uL of the standards.
- 20 uL of the lavage samples were added to adjacent rows in the
well plate.
- 100 uL of the Coomassie Plus reagent was added for a final volume
of 120 uL.
- Samples/standards incubated for 5 minutes.
- Absorbance was read at 620nm.
Immunoglobulin A (IgA) assay:
- In order to measure the amount of IgA in the human lung fluid
samples, an ELISA was ran similar to that of the Albumin ELISA
using the same set of samples.
- For the standard curve, several serial dilutions were made from
the 2.5mg/mL stock IgA solution. 5uL of IgA stock with was diluted
to 10ng/uL and serially diluted to 5, 2.5, 1.25, .63, .32, and
.16, .08ng/uL.
- The primary and secondary antibodies used included the Antihuman
IgA alpha chain specific peroxidase conjugate developed in goat
and Antigoat IgG whole molecule peroxidase conjugate developed
in rabbit, respectively. Also incubation times were slightly shorter
than the Albumin ELISA
- To compensate for variability in dilution by the lavage procedure,
the H2O2 and albumin measurements were normalized to the IgA measurements
obtained from the samples.
Statistical Analysis:
- Normalization data was analyzed using Sigma Plot software. Comparisons
were made using the Kruskal-Wallis One Way Analysis of Variance
on Ranks and the T-Test. Statistical significance was accepted
at a p-value < or = 0.05.
Figure
1
- Oxidative stress in the lung was greater in otherwise healthy
alcoholics than control subjects as assessed by H2O2.
- Oxidative stress trended to be greater in alcoholics than smokers
but more subjects are needed.
- Oxidative stress improved after 1 week of abstinence.
Figure
2
- In ARDS patients the greater oxidative stress in alcoholics
is approaching statistical significance.
Figure
3
- Albumin leak was greater in otherwise healthy alcoholics when
compared to control and smoking subjects.
- Albumin leak trended to improve after 1 week of abstinence from
alcohol ingestion. More subjects are needed.
Figure 4
- In ARDS patients, a history of alcohol abuse did not result
in greater albumin leak.
Figure
5
- There was a positive correlation between H2O2 in the lavage
and albumin leak, a marker of lung injury. H2O2 is also present
in the exhaled breath. Therefore measurements of H2O2 in a breath
condensate may provide a simple and non-invasive tool to assess
lung injury.
Lou Ann S. Brown, PhD., Husni Elbahesh, Frank Harris,
Xiao Du Ping, Michael Wong Marc Moss, M.D. Ellen Burnham, M.D. Illustration
by Kombo Chapfika. This research was funded by: Howard Hughes Medical
Institute (Grant No. 52003071) and the National Institute of Health
(Grant No. 1 RO3 HL67399 & 1 RO1 AA 12197)
Our research deals with assessing lung damage by measuring
the amounts of hydrogen peroxide and albmunin protein in the lung
fluid of alcoholics, smokers and people that suffer from Acute Respiratory
distress syndrome. In this study we found that excessive ingestion
of alcohol can lead to an increase in hydrogen peroxide and albumin
in the lung fluid. Our data showed that there was more H2O2 in alcoholic
patients with ARDS than in the control ARDS patients. We also found
that after one week of not drinking the levels of H2O2 and albumin
decreased. Levels of the oxidant H2O2 were higher in the alcoholics
than in the smokers, but this was not the case with alubumin. In
the end more patient samples need to be collected in order to further
verify this data.
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