Shelley S. Sehnert

Clinical application of breath biomarkers of oxidative stress status

Isolation and quantification of volatile breath biomarkers indicative of relevant alterations in clinical status has required development of new techniques and applications of existing analytical chemical methods. The most significant obstacles to successful application of this type of sample have been reduction in required sample volume permitting replicate analysis (an absolute requirement for all clinical studies), separation of the analyte(s) of interest from background molecules, water vapor and other molecules with similar physical properties, introduction of automation in analysis and the use of selective detection systems (electron impact mass spectrometry, flame photometric, thermionic detectors), and automated sample collection from the human subject. Advances in adsorption technology and trace gas analysis have permitted rapid progress in this area of clinical chemistry.

Breath biomarkers for detection of human liver diseases: preliminary study

Chronic liver disease is initially occult, has multiple aetiologies, involves complex diagnostic questions, and requires follow-up because progression is likely. Blood tests and biopsies are generally used, but have disadvantages. We have developed a new test for liver disease based on abnormal concentrations of metabolic products detected in exhaled breath. This test can be used, in conjunction with other clinically accepted diagnostic protocols, to detect and classify chronic liver diseases. Samples of breath collected from spontaneously breathing human subjects (86 patients presenting with 13 liver diseases and 109 subjects with normal liver function) were concentrated cryogenically and analysed by wide-bore capillary gas chromatography using various detectors. The concentrations of various molecules in exhaled breath were examined for potential use as biomarkers of liver function. Subjects with chronic liver diseases could be differentiated from those with normal liver function by comparing levels of breath carbonyl sulphide, carbon disulphide and isoprene; these differences were confirmed and correlated by comparing the levels with standard clinical blood markers of liver damage. The presence of chronic liver failure can thus be detected with sensitivity and specificity by quantifying sulphur-containing compounds arising from the abnormal metabolism associated with liver disease. The breath test we have developed appears to distinguish between hepatocellular and biliary tract aetiologies, and allows staging for severity. This approach may provide the clinician with a simple, non-invasive technique for use in the screening of large populations and follow-up for patients with chronic liver disease.

Breath ethane generation during clinical total body irradiation as a marker of oxygen-free-radical-mediated lipid peroxidation: a case study.

Total body irradiation (TBI) is used therapeutically for treatment of leukemias and other malignancies of the hemopoietic system. Ionizing radiation produces oxygen free radicals that contribute to cytotoxicity. Breath collected from one patient undergoing therapeutic TBI showed measurable changes in levels of ethane during treatment. Breath ethane is a marker of lipid peroxidation of n-3 fatty acids. The TBI treatment involved 4 days of irradiation. The largest changes in breath ethane occurred on Day 2. The increased levels of breath ethane on Day 2 were correlated to clinical manifestations of toxicity. The correlation of the onset of gastrointestinal side effects with higher levels of breath ethane suggests that breath ethane may be a clinically useful measure of the toxicity of various TBI fractionation treatment protocols currently in use at different medical centers. The levels of breath ethane on the other days of treatment were lower, suggesting that the oxidative-antioxidative balance of the patient may be important in protection against free radical mediated injury. These results for a single patient suggest that breath ethane may be a promising approach to elucidate the role of antioxidants in clinical TBI and should be extended for verification to a larger volunteer patient population.

Breath ethane: a specific indicator of free-radical-mediated lipid peroxidation following reperfusion of the ischemic liver.

A major component of the organ injury mediated by toxic oxidants, such as seen following reperfusion of the ischemic liver, is due to the peroxidation of polyunsaturated fatty acids, especially of cell membranes. We utilized the measurement of exhaled breath ethane, a metabolic product unique to oxidant-mediated lipid peroxidation, as a noninvasive indicator of this process in swine liver subjected to warm ischemia/reperfusion. Under rigorously controlled anesthesia conditions, pig livers were subjected to 2 h of warm total ischemia, followed by reperfusion in situ. Expired air was collected and its ethane content quantitated by a novel gas chromatographic technique. The time course of breath ethane generation correlated closely with the appearance of hepatocellular injury as measured by impairment of Factor VII generation and other measures of liver integrity. Moreover, the administration of the specific superoxide free radical scavenger, superoxide dismutase (SOD), significantly attenuated both the elaboration of ethane and the hepatocellular injury. These findings not only provide confirmation of the previously reported link between hepatocellular injury by free radicals generated at reperfusion, but also establish the use of expired breath ethane analysis as a sensitive, specific, and noninvasive indicator of the injury process in real time.

Visceral lipid peroxidation occurs at reperfusion after supraceliac aortic cross-clamping

PURPOSE Recently, we have reported that lipid peroxidation specific to oxygen free radical-mediated injury increased immediately after reperfusion of human liver allografts. However, in the human liver transplantation setting it was impossible to disassociate the contributions to lipid peroxidation caused by the warm and cold ischemic phases from those caused by reperfusion. Therefore we now have studied lipid peroxidation at reperfusion after supraceliac aortic cross-clamping in patients with normal livers. METHODS Ethane, a noninvasive biomaker of lipid peroxidation, was measured in exhaled breath of patients before and during cross-clamping of the thoracic aorta and at sequential time intervals after visceral reperfusion. RESULTS Approximately a two-fold transient increase in the ethane level was observed at around 15 minutes after reperfusion in those patients whose aortas were cross-clamped for more than 18 minutes. CONCLUSIONS These results indicate that free radical-mediated lipid peroxidation occurs at reperfusion of warm ischemic viscera in the clinical setting of aortic repair. This observation supports the hypothesis that substantial lipid peroxidation occurs when tissues are subjected to cold or warm ischemia followed by reperfusion.

ETHANE: A MARKER OF LIPID PEROXIDATION DURING CARDIOPULMONARY BYPASS IN HUMANS

The goals of this study were to (1) determine the utility of quantification of ethane as a marker of ischemia-reperfusion during human cardiopulmonary bypass (CPB); and (2) determine, using an animal model for this surgical procedure, whether the mode of surgical approach produced increases the quantity of exhaled ethane. Human CPB was initiated following standard anesthetic and monitoring regimens. Samples of gas were collected at baseline and at multiple defined time points throughout the studies. Ethane was determined using cryogenic concentration and gas chromatography. Sternotomy increased exhaled ethane compared to baseline (p < .007; 5.8 +/- 1.7 vs. 3.0 +/- 0.7 nmol/m2 x min); ethane returned to baseline levels prior to the initiation of CPB. Aortic unclamping produced ethane elevation (p < .05; 2.3 +/- 0.8 vs. 1.5 +/- 0.4 nmol/m2 x min) with the levels being related to a lower cardiac index and a higher systemic vascular resistance post aortic unclamping. Termination of CPB significantly increased ethane levels compared to baseline (p < .002; 4.8 +/- 1.7 vs. 3.0 +/- 0.7 nmol/m2 x min). Independent variables that correlated with increased ethane measurements included a higher arterial blood pH on bypass and the change in hemoglobin pre- and post-CPB. Electrocautery, but not scalpel, incision of the porcine abdominal wall increased ethane levels significantly (p < .02). These results indicate that exhaled ethane may be a valuable marker of lipid peroxidation during and following CPB.

Prooxidant effects of maternal smoking and formula in newborn infants

BACKGROUND The purpose of this study was to use the breath ethane test to determine if either maternal cigarette smoking, formula, and/or deficiency of the antioxidant nutrients vitamins A and E was associated with oxidant stress in newborn infants. The rationale for this study was: (1) our observation that cigarette smoking was a source of oxidant stress in pregnant women, suggesting that it could be a source of oxidant stress for infants exposed in utero; (2) formula was predicted to be prooxidant compared to colostrum, which contains several compounds with antioxidant activity in vitro; and (3) deficiencies of vitamins A and E have been shown to promote oxidant stress in experimental animals. METHODS Breath ethane, a volatile alkane produced by peroxide of n-3 fatty acids, was utilized as an index of oxidant stress status. Forty-five healthy full-term infants of the women mentioned above were studied at 18-24 h of age, after four to six feedings of breast milk (colostrum) or caseinbased infant formula. Relationships between infant breath ethane, maternal smoking, mode of infant nutrition, and serum concentrations of the antioxidant vitamins A and E of infants were examined. RESULTS The breath ethane of the entire group of infants whose mothers smoked (n = 19) was increased compared to values of infants whose mothers did not smoke (n = 26): 97 +/- 16 versus 43 +/- 9 pmol/kg/min, p < 0.03. When infants of mothers who smoked were eliminated from the analysis in order to study effects of nutrition alone, formula appeared to be prooxidant compared to breast milk. Breath ethane of formula-fed infants (n = 16) was 62 +/- 13 versus 13 +/- 4 pmol/kg/min for breast-fed infants (n = 10), p < 0.04. For the group as a whole, there was no correlation between infant breath ethane and serum concentrations of vitamins A and E. CONCLUSIONS Exposure to maternal smoking in utero is prooxidant in newborn infants. Formula also has a prooxidant effect compared to colostrum in newborn infants not exposed to maternal smoking in utero. Further investigations will be necessary to explore the clinical consequences of these observations.

Possible antioxidant effect of vitamin A supplementation in premature infants

BACKGROUND Increased lipid peroxidation caused by oxygen free radicals is thought to be one of the common pathogenetic mechanisms for the so-called oxygen radical diseases of prematurity. Since in vitro studies have shown that various forms of vitamin A can exert antioxidant effects that are more potent than those of vitamin E (treatment with which has been ineffective in these diseases), the purpose of this prospective, controlled study was to determine whether administration of supplemental vitamin A to premature infants deficient in this vitamin would have an antioxidant effect in vivo. METHODS Fourteen infants (1181 +/- 35 g; gestational age 29 +/- 0.04 weeks) with a serum retinol concentration at 7 +/- 2 days of age in the deficient range, lower than 0.7 mumol/l (< 20 micrograms/dl), were enrolled in the study. Infants were randomized to receive the standard amount of vitamin A or standard plus supplemental (2.6 mumol/l [2500 IU] orally each day) vitamin A, beginning at 1 week of age. Antioxidant effects of supplementation were assessed by a decrease in lipid peroxidation, quantified by the ethane content of expired air. RESULTS Three weeks after study enrollment, total daily vitamin A intake in the infants receiving supplements was 4.565 +/- 0.236 mumol (4354 +/- 225 IU) versus 1.879 +/- 0.317 mumol/l (1792 +/- 302 IU) in infants receiving standard amounts of the vitamin. In spite of the difference in intake of vitamin A, 3 weeks after study enrollment, serum retinol concentrations did not differ between the infants given supplements and those receiving standard amounts of vitamin A, 0.70 +/- 0.21 versus 0.66 +/- 0.07 mumol/l (20 +/- 6 micrograms/dl versus 19 +/- 2 micrograms/dl, respectively). In the infants receiving supplemental vitamin A, breath ethane values declined from baseline values. There was an inverse correlation between the number of weeks of supplementation and breath ethane values, whereas there was no significant correlation between the duration of the study and breath ethane values in the infants not given supplements. CONCLUSIONS Our data suggest that supplementation with vitamin A in a small group of vitamin A-deficient preterm infants was associated with an antioxidant effect. Although no immediate clinical benefits were associated with supplementation, the data provide the rationale for future investigations of possible antioxidant effects of (larger amounts?) of vitamin A in higher risk premature infants born with subnormal serum retinol concentrations.

Cigarette Smoking Is Pro-oxidant in Pregnant Women Regardless of Antioxidant Nutrient Intake

Oxygen radical injury may be a common pathogenetic mechanism in several diseases of newborn infants; the objective of our study was to investigate nutritional and environmental factors which contribute to oxidative stress during pregnancy. Ethane, a volatile alkane produced during peroxidation of omega-3 fatty acids, was used as a non-invasive indicator of oxidative stress. Forty-four inner-city pregnant women were studied to investigate relationships between breath ethane, antioxidant nutritional status and the use of drugs, alcohol and/or tobacco. The mean age of the women was 24 ± 1 years; they were studied at 28 weeks of gestation. Nineteen of the 44 women (43%) smoked, five (11%) had positive urine drug screens and three (7%) consumed alcoholic beverages. No relationship was shown between the breath ethane of the whole group and intake of total calories, fat, protein or vitamins A, C, E, carotene or iron. Similarly, there were no correlations between breath ethane and serum vitamins A, E, E/total lip...

Nose-only inhalation system using the fluidized-bed generation system for coexposures to carbon black and formaldehyde

AbstractThe purpose of this research was to develop a nose-only inhalation toxicology system for exposing animals to carbon black and formaldehyde—both individually and in combination. This study discusses the modifications necessary to use a fluidized-bed generator to generate carbon black in the concentration range of 0.5–20 mg/m3 with a mass median aerodynamic diameter of approximately 2.5 μm. The specific recommendations involve changing the size and placement of the delivery and overflow tubes as well as installing a motorized ball valve to automate delivery of premixed bed material and carbon black to the generator. The inhalation chamber was modified from a previously published design. By using a cyclone preclassifier to remove the nonrespirable fraction of the aerosol and a laminar flow element to reduce inlet vortic-ity, it was possible to obtain a uniform chamber concentration with less than ±25% overall variation. The formaldehyde generation system produced stable concentrations in the range of...