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.

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.

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.

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.

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.

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.

Evidence for free radical-mediated lipid peroxidation at reperfusion of human orthotopic liver transplants.

BACKGROUND: Generation of toxic oxygen metabolites at reperfusion may contribute to the injury sustained as a consequence of harvest and ischemic preservation of organ allografts. Because there is a paucity of evidence that this mechanism is operative in human beings, we measured the generation of ethane into the exhaled breath as a biomarker of free radical-mediated lipid peroxidation in human liver transplantation. METHODS: A novel technique that increased the previous standard of sensitivity 100-fold was used to measure picomole quantities of ethane in exhaled breath of eight recipients undergoing human orthotopic liver transplantation. RESULTS: Ethane production correlated closely with the specific events of liver transplantation including the initial reperfusion of the allografts. In every case a twofold to threefold increase in ethane production was superimposed on a stable baseline immediately after reestablishment of portal vein blood flow through the donor liver. CONCLUSIONS: Ethane production was interpreted as evidence of hepatic lipid peroxidation, presumably mediated by toxic metabolites of oxygen occurring at reperfusion. This noninvasive approach allowed localization of the time point at which lipid peroxidation occurred and may facilitate quantification of lipid peroxidation mediated by free radicals and other toxic oxygen metabolites during operation.

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.

Suppression of alveolar macrophage membrane-receptor-mediated phagocytosis by model particle-adsorbate complexes: physicochemical moderators of uptake.

In order to assess the abilities of alveolar macrophages (AMs) to phagocytize adsorbent-adsorbate complexes, rat AMs were incubated in vitro with two carbon blacks that have 15-fold differences in specific surface areas (ASTM classification N339 less than Black Pearls 2000) sorbed with 0.5 and 1.0 monolayer coverages of a polar and semi-polar adsorbate (acrolein and benzofuran, respectively). One-half monolayer coverages of N339 with either adsorbates significantly suppressed the phagocytosis of the carbon black, whereas one monolayer coverage did not. Neither adsorbate at either coverages affected the phagocytosis of Black Pearls 2000. The capacity of macrophages to phagocytize a subsequent particle challenge via the Fc-membrane receptor was quantified following treatment of the macrophages with the carbon black-adsorbate complexes. Treatment of the macrophages with carbon black N339-adsorbates complexes at both coverages impaired Fc-receptor-mediated phagocytosis, whereas no effect was observed when the carbon black was Black Pearls 2000. The results of this study indicate that the surface properties of the particles, the chemical properties of the chemical pollutants, and the interactions between particles and pollutants play a major role in defining the biological effect of particle-pollutant complexes.

Suppression of alveolar macrophage membrane receptor-mediated phagocytosis by model and actual particle-adsorbate complexes. Initial contact with the alveolar macrophage membrane.

Alveolar macrophages were treated with carbon blacks and adsorbates in order to evaluate the biologic effect of adsorbate, adsorbent and adsorbate-adsorbent complexes. Their capacity to phagocytize a subsequent challenge via the Fc-membrane receptor was quantified. Phagocytosis was suppressed in a dose-related manner with increasing concentrations of both carbon blacks and adsorbates. Carbon black N339 covered with 0.5 monolayers of the adsorbates suppressed phagocytosis more than N339 without the adsorbates. Increasing the adsorbate acrolein coverage from 0.5 to greater than 2.0 monolayers suppressed phagocytosis in a dose-related manner. Finally, samples of diesel particulate matter collected from an engine operated on a pure hydrocarbon fuel with various oxidizers, air (PSU #1) and an oxidizer free of nitrogen (N-free) were tested. Treatment of the macrophages with PSU #1 had a negligible effect on phagocytosis whereas the N-free sample suppressed phagocytosis in a dose-related manner. The data show that alveolar macrophage Fc-receptor-mediated phagocytosis is affected by: carbon black and adsorbate identity and concentration, coverage of the carbon black with adsorbates, and the oxidizer used in the generation of particles emitted by a diesel engine.

Chromatographic modeling of the release of particle-adsorbed molecules into synthetic alveolar surfactant.

Pseudophase liquid chromatography was used to measure the thermodynamic parameters governing adsorption of organic molecules from the surfaces of carbonaceous particles into liposomal zwitterionic mobile phases. These mobile phases contain many of the important physicochemical parameters of alveolar surfactant. Results show that physical desorption into model surfactant will be dependent upon the heat of solution and the heat of adsorption. Dominance of either thermodynamic parameter is dependent upon the relative polarity of the adsorbent surface and the adsorbate molecule. It is postulated from data obtained from simple molecules containing relevant organic functional groups that physical desorption of environmental agents from the surfaces of particulate complexes into alveolar surfactant may be predicted both by quantification of the polarity of the system and of the extent of surface coverage under investigation.

A model for the formation of airborne particulate matter based on the gas-phase adsorption on amorphous carbon blacks.

This paper reports the physicochemical properties that describe the adsorption of a series of solutes onto the surfaces of amorphous carbon blacks. Adsorption was studied at concentrations that correspond to low surface coverages and in the presence of volatile solvent diluents. The adsorbates and adsorbents were selected for their relevance as models for environmental agent-particle complexes originating from incomplete combustion. The data clearly show that the major factors that determine the strength of adsorption are the surface properties of the adsorbent and the intermolecular forces between the surface and the adsorbing molecule. The heat of adsorption data have been used to predict the lifetime of the absorbate-adsorbent complexes.

A model for the release of adsorbed molecules from the surfaces of airborne particulate matter based on liquid-phase desorption from amorphous carbon blacks.

The release of molecules adsorbed on the surfaces of amorphous carbon blacks has been studied using liquid-solid chromatography. Adsorbate molecules, adsorbents, and mobile phases were selected on the basis of their relevance as models for the release of toxic agents adsorbed on inhalable environmental particulate matter that originates from the incomplete combustion of organic materials. The presence of surface active groups on the carbon blacks has been shown to adsorb and retain adsorbate molecules selectively, and this selectivity can be reduced by competition for these active groups by the displacing solvent. Release is also governed by the surface coverage of the particles and increases as coverage approaches the monolayer.