Thiol redox status in critically ill patients Open Access

Wheaton, Anne-Genevieve (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/hx11xf95h?locale=en
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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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