Abstract
Thiol Redox Status in Critically Ill Patients
By Anne-Genevieve Wheaton
Oxidative stress has been implicated in many pulmonary diseases.
Previous studies have focused on the status
of the endogenous antioxidant glutathione (GSH), but the status of
cysteine (Cys), another thiol
antioxidant present in the lung, has been largely ignored. The
bronchoalveolar lavage fluid of
critically ill patients was more oxidized than controls as
evidenced by higher H2O2,
4-hydroxynonenal,
and %GSSG (100%*GSSG/GSH+GSSG). The Cys pool was larger in this
group but not significantly more
oxidized than controls. Higher malondialdehyde concentrations and
%GSSG in critically ill patients
indicated more oxidized plasma, but surprisingly plasma Cys was
more reduced in this group. A history of
alcohol abuse is a risk factor for the development of and increased
mortality from ventilator-associated
pneumonia (VAP), a common infection among mechanically-ventilated
patients. GSH was depleted to a
comparable extent in healthy alcoholics and VAP patients compared
to healthy controls. The GSH pools were
also more oxidized in these groups. Cys and CySS (cystine)
concentrations were much higher in
the VAP groups than the healthy groups. The total Cys pool (tCys)
was larger than the GSH pool
(tGSH) in VAP patients compared to healthy subjects. Among VAP
patients, lower tCys/tGSH was
associated with survival. Although smoking and chronic alcohol
abuse are both oxidative conditions, each is
associated with increased risk for different pulmonary disorders.
The exhaled breath condensate (EBC), a
noninvasive sampling method, of subjects with a history of alcohol
abuse and/or smokers was used to monitor
the redox status of GSH and Cys. GSH was more oxidized in the EBC
of alcoholics, but not different based
on smoking status. Alcohol abuse was associated with elevated Cys
and CySS concentrations in
the EBC. Smoking was associated with a 50% decrease in the Cys
concentration. Cys was
more oxidized by smoking in the control groups and by alcohol in
the nonsmoking groups. The ratio of
tCys/tGSH was approximately one in nonsmoking controls. This ratio
was significantly higher for both
alcohol groups and lower for smoking controls, possibly indicative
of different forms of oxidative stress.
Table of Contents
TABLE OF CONTENTS
Chapter 1:
Introduction...................................................................................................
1
1.1 Clinical
Significance
.................................................................................................
1
1.1.1 Acute Respiratory Distress
Syndrome.......................................................
1
1.1.2 Ventilator-Associated
Pneumonia
............................................................
4
1.1.3 Alcohol
Abuse.........................................................................................
4
1.1.4
Smoking.................................................................................................
5
1.2 Oxidative
Stress
......................................................................................................
6
1.2.1 What is Oxidative
Stress?........................................................................
6
1.2.2 Redox
Signaling......................................................................................
7
1.2.3 Important Pro-Oxidants in the
Lung..........................................................
7
1.2.4
Antioxidants.........................................................................................
10
1.2.5 Sources of Oxidants in the
Lung..............................................................
21
1.3 The Pulmonary
Milieu
...............................................................................................
24
1.3.1 The "Conducting
Zone"..........................................................................
24
1.3.2 Gas Exchange
Zone...............................................................................
27
1.3.3 Pulmonary
Defenses.............................................................................
27
1.4 Sampling Methodologies for Assessing Oxidative
Stress
..................................................
28
1.4.1 Bronchoalveolar
Lavage........................................................................
28
1.4.2
Plasma.................................................................................................
30
1.4.3 Exhaled Breath
Condensate...................................................................
31
1.5 The
Studies
...........................................................................................................
32
1.5.1 Altered Pulmonary and Systemic
Thiols in Critically Ill Patients (Chapter
3).32
1.5.2 Thiols in the Bronchoalveolar
Lavage Fluid of Ventilator-Associated Pneumonia Patients with and
without Alcohol Abuse (Chapter
4)..................................................................................
33
1.5.3 Divergent Effects of Alcohol
and Smoking on Thiols in Exhaled Breath Condensate (Chapter
5)35
Chapter 2:
Methods...............................................................................................................
37
2.1 Subject
Recruitment
..................................................................................................
37
2.1.1 Healthy
Subjects...................................................................................
37
2.1.2 Alcohol Abuse
Status.............................................................................
37
2.2 Screening Tests for Alcohol
Abuse
................................................................................
38
2.2.1 Short Michigan Alcohol
Screening
Test.....................................................
38
2.2.2 Alcohol Use Disorders
Identification
Test.................................................
40
2.3 Sample
Collection
.......................................................................................................
40
2.3.1 Bronchoalveolar
Lavage........................................................................
40
2.3.2
Plasma.................................................................................................
42
2.3.3 Exhaled Breath
Condensate...................................................................
42
2.4 Measurement of
Thiols
................................................................................................
42
2.4.1 Sample
Preservation.............................................................................
43
2.4.2 Derivatization of
Samples......................................................................
43
2.4.3 High Performance Liquid
Chromatography..............................................
44
2.5 Measurement of
Oxidants
............................................................................................
45
2.5.1 Hydrogen
Peroxide................................................................................
45
2.5.2 4-Hydroxynonenal
(HNE).......................................................................
45
2.5.3 Malondialdehyde
(MDA).........................................................................
45
Chapter 3: Altered Pulmonary and Systemic Thiols in
Critically Ill Patients...............................
47
3.1
Background
...............................................................................................................
47
3.2
Methods
...................................................................................................................
48
3.2.1 Subject
Recruitment..............................................................................
48
3.2.2 Sample
Collection.................................................................................
49
3.2.3 Markers of Oxidative
Stress....................................................................
49
3.2.4 HPLC Measurements of
Thiols.................................................................
50
3.2.5 Statistical
Analysis................................................................................
51
3.3
Results
......................................................................................................................
51
3.3.1 Subject
Demographics...........................................................................
51
3.3.2 BALF H2O2
and HNE were Higher in the Critically Ill
Patients...................... 51
3.3.3 Lower GSH Concentrations and
Higher %GSSG in Patient BALF..................
55
3.3.4 Increased BALF Cys and CySS
Concentrations in the Critically Ill...............
55
3.3.5 Associations between BALF
Glutathione and Cysteine..............................
60
3.3.6 Correlations Between Thiols and
Oxidative Stress Markers in the BALF.......60
3.3.7 Lipid Peroxidation in Plasma of
Patients..................................................
60
3.3.8 Increased Plasma GSSG and tGSH
Concentrations, and %GSSG in Critically Ill. 65
3.3.9 Increased Plasma Cys and tCys,
but Lower %CySS in Critically Ill.............
65
3.3.10 Associations between Plasma
Glutathione and Cysteine.............................
65
3.3.11 Correlations between Thiols
and Oxidative Stress Markers in Plasma.
..........70
3.3.12 Correlations of Thiols in BALF
and
Plasma..................................................
70
3.4
Discussion
....................................................................................................................
70
Chapter 4: Thiols in the Bronchoalveolar Lavage Fluid
of Ventilator-Associated Pneumonia Patients with and without
Alcohol
Abuse............................................................................................................................78
4.1
Background
...................................................................................................................
78
4.2
Methods
.......................................................................................................................
80
4.2.1 Subject
Recruitment..............................................................................
80
4.2.2 Sample
Collection.................................................................................
81
4.2.3 Markers of Oxidative
Stress...................................................................
82
4.2.4 HPLC Measurements of
Thiols.................................................................
83
4.2.5 Statistical
Analysis................................................................................
83
4.3
Results
...........................................................................................................................
83
4.3.1 Subject
Demographics............................................................................
83
4.3.2 Oxidative Stress Markers were
increased in the BALF of VAP Patients..........85
4.3.3 GSH Concentrations in the BALF
of Healthy Alcoholics were Comparable to VAP Patients
85
4.3.4 BALF GSH was More Oxidized in
Healthy Alcoholics and VAP Patients...........90
4.3.5 Cys Concentrations in the BALF
of VAP Patients were Higher Than in Healthy Subjects
90
4.3.6 BALF Cys was Oxidized in
Healthy
Alcoholics..............................................95
4.3.7 The Cys Pool (tCys) in VAP
Patients was Larger than the GSH Pool (tGSH)....
95
4.3.8 Survival Corresponded with a
Lower tCys/tGSH
Ratio..................................95
4.4
Discussion
......................................................................................................................
102
Chapter 5: Divergent Effects of Alcohol and Smoking on
Thiols in Exhaled Breath
Condensate................................................................................................................................
108
5.1
Background
....................................................................................................................
108
5.2
Methods
........................................................................................................................
111
5.2.1
Subjects................................................................................................
111
5.2.2 Sample
Collection..................................................................................
111
5.2.3 HPLC Measurement of
Thiols...................................................................
112
5.2.4 Statistical
Analysis.................................................................................
112
5.3
Results
...........................................................................................................................
113
5.3.1 Subject
demographics.............................................................................
113
5.3.2 Alcohol Abuse was Associated
with a Decrease in GSH and an Increase in GSSG113
5.3.3 Alcohol Abuse was Associated
with an Oxidation of GSSG...........................
113
5.3.4 Alcohol Abuse was Associated
with an Increase in Cys and Smoking with a Decrease in Cys
.. 115
5.3.5 Alcohol Abuse was Associated
with an Increase in CySS.............................
115
5.3.6 Cys was More Oxidized in
Alcoholics and
Smokers.....................................
120
5.3.7 The Ratio of Total Cysteine to
Total Glutathione was Increased with Alcohol Abuse, but Decreased
with
Smoking...............................................................................................................
120
5.4
Discussion
.......................................................................................................................
127
Chapter 6:
Discussion.......................................................................................................................
132
6.1 Altered Thiol Status in Critically Ill
Patients
.............................................................................
132
6.1.1 Possible Mechanisms for
Alteration of Thiol
Status......................................
132
6.1.2 Future
Directions.....................................................................................
134
6.2 Comparable Oxidation of Bronchoalveolar Lavage Fluid GSH
in Healthy Alcohol Abusers and VAP
patients
...................................................................................................................................
135
6.3 Chronic Alcohol Abuse History Associated with Oxidized GSH
but Reduced Cys in Exhaled Breath
Condensate
...............................................................................................................................
136
References.......................................................................................................................................
138
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