Utilization of a highly adaptable murine air pouch model for minimally invasive testing of the inflammatory potential of biomaterials.

Introduction: The biocompatibility of an implanted material strongly determines the subsequent host immune response. After insertion into the body, each medical device causes tissue reactions. How intense and long-lasting these are is defined by the material properties. The so-called foreign body reaction is a reaction leading to the inflammation and wound healing process after implantation. The constantly expanding field of implant technology and the growing areas of application make optimization and adaptation of the materials used inevitable. Methods: In this study, modified liquid silicone rubber (LSR) and two of the most commonly used thermoplastic polyurethanes (TPU) were compared in terms of induced inflammatory response in the body. We evaluated the production of inflammatory cytokines, infiltration of inflammatory cells and encapsulation of foreign bodies in a subcutaneous air-pouch model in mice. In this model, the material is applied in a minimally invasive procedure via a cannula and in one piece, which allows material testing without destroying or crushing the material and thus studying an intact implant surface. The study design includes short-term (6 h) and long-term (10 days) analysis of the host response to the implanted materials. Air-pouch-infiltrating cells were determined by flow cytometry after 6 h and 10 days. Inflammation, fibrosis and angiogenesis markers were analyzed in the capsular tissue by qPCR after 10 days. Results: The foreign body reaction was investigated by macroscopic evaluation and scanning electron microscopy (SEM). Increased leukocyte infiltration was observed in the air-pouch after 6 h, but it markedly diminished after 10 days. After 10 days, capsule formations were observed around the materials without visible inflammatory cells. Discussion: For biocompatibility testing materials are often implanted in muscle tissue. These test methods are not sufficiently conclusive, especially for materials that are intended to come into contact with blood. Our study primarily shows that the presented model is a highly adaptable and minimally invasive test system to test the inflammatory potential of and foreign body reaction to candidate materials and offers more precise analysis options by means of flow cytometry.

Development of a Novel Valve-Controlled Drug-Elutable Microstent for Microinvasive Glaucoma Surgery: In Vitro and Preclinical In Vivo Studies.

Purpose: Microinvasive glaucoma surgery (MIGS) has become an important treatment approach for primary open-angle glaucoma, although the safe and long-term effective lowering of intraocular pressure with currently available implants for MIGS is not yet achieved to a satisfactory extent. The study focusses on the development and in vitro and in vivo testing of a novel microstent for MIGS. Methods: A silicone elastomer-based microstent was developed. Implants were manufactured using dip coating, fs-laser cutting, and spray coating. Within the current study no antifibrotic drug was loaded into the device. Sterilized microstents were analyzed in vitro regarding pressure-flow characteristics and biocompatibility. Six New Zealand white rabbits were implanted with a microstent draining the aqueous humor from the anterior chamber into the subconjunctival space. Drainage efficacy was evaluated using oculopressure tonometry as a transient glaucoma model. Noninvasive imaging was performed. Results: Microstents were manufactured successfully and characterized in vitro. Implantation in vivo was successful for four animals with additional device fixation. Without additional fixation, dislocation of microstents was found in two animals. Safe and effective intraocular pressure reduction was observed for the four eyes with correctly implanted microstent during the 6-month trial period. Conclusions: The described microstent represents an innovative treatment approach for MIGS. The incorporation of a selectively antifibrotic drug into the microstent drug-elutable coating will be addressed in future investigations. Translational Relevance: The current preclinical study successfully provided proof of concept for our microstent for MIGS which is suitable for safe and effective intraocular pressure reduction and offers promising perspectives for the clinical management of glaucoma.

Vascular Response Toward an Absorbable Sirolimus-eluting Polymeric Scaffold for Vascular Application in a Model of Normal Porcine Carotid Arteries.

BACKGROUND: Fully absorbable polymeric scaffolds, as a potential alternative to permanent metallic stents, are entering the clinical field. The aim of this study is to assess the in vivo biocompatibility of a novel Sirolimus-eluting (SIR) absorbable scaffold based on poly(L-lactide) (PLLA) and poly(4-hydroxybutyrate) (P4HB) for interventional application. METHODS: Absorbable PLLA/P4HB scaffolds either loaded with SIR coating or unloaded scaffolds were implanted interventionally into common carotid arteries of 14 female. Bare metal stents (BMS) served as control. Peroral dual anti-platelet therapy was administered throughout the study. Stented common carotid arteries segments were explanted after 4 weeks, and assessed histomorphometrically. RESULTS: The absorbable scaffolds showed a decreased residual lumen area and higher stenosis after 4 weeks (PLLA/P4HB: 6.56 ± 0.41 mm² and 37.56 ± 4.67%; SIR-PLLA/P4HB: 6.90 ± 0.58 mm² and 35.60 ± 3.15%) as compared to BMS (15.29 ± 1.86 mm² and 7.65 ± 2.27%). Incorporation of SIR reduced the significantly higher inflammation of unloaded scaffolds however not to a level compared to bare metal stent (PLLA/P4HB: 1.20 ± 0.19; SIR-PLLA/P4HB: 0.96 ± 0.24; BMS: 0.54 ± 0.12). In contrast, the BMS showed a slightly elevated vascular injury score (0.74 ± 0.15), as compared to the PLLA/P4HB (0.54 ± 0.20) and the SIR-PLLA/P4HB (0.48 ± 0.15) groups. CONCLUSION: In this preclinical model, the new absorbable polymeric (SIR-) scaffolds showed similar technical feasability and safety for vascular application as the permanent metal stents. The higher inflammatory propensity of the polymeric scaffolds was slightly reduced by SIR-coating. A smaller strut thickness of the polymeric scaffolds might have been a positive effect on tissue ingrowth between the struts and needs to be addressed in future work on the stent design.

Evaluation of a Murine Model for Testing Antimicrobial Implant Materials in the Blood Circulation System.

Medical device-related infections are becoming a steadily increasing challenge for the health care system regarding the difficulties in the clinical treatment. In particular, cardiovascular implant infections, catheter-related infections, as well as infective endocarditis are associated with high morbidity and mortality risks for the patients. Antimicrobial materials may help to prevent medical device-associated infections and supplement the currently available therapies. In this study, we present an easy-to-handle and simplified in vivo model to test antimicrobial materials in the bloodstream of mice. The model system is composed of the implantation of a bacteria-laden micro-stent scaffold into the murine tail vein. Our model enables the simulation of catheter-related infections as well as the development of infective endocarditis specific pathologies in combination with material testing. Furthermore, this in vivo model can cover two phases of the biofilm formation, including both the local tissue response to the bacterial biofilm and the systemic inflammatory response against circulating bacteria in the bloodstream that detached from a mature biofilm.

A preclinical animal model for evaluating the sealing capacity of covered stent grafts in acute vessel perforation.

BACKGROUND: Percutaneous coronary intervention is among the most common therapeutic interventions in cardiology. This procedure may, however, be associated with a rare, though life-threatening complication: acute coronary perforation (CP). CP is primarily treated using covered stents, which are made of bare metal stents with a polytetrafluoroethylene (PTFE) or polyurethane coating. These stents' major limitations include higher rates of thrombus formation and restenosis. Hence, there is a still unmet need for new stents regarding their design and composition. Or, to test new covered stent designs, the rabbit iliac artery has become the best-established animal model. This study sought to present a preclinical animal approach designed to test covered stents that are utilized following vessel perforation. METHODS: The animal experiments were performed using New Zealand white rabbits, each weighting 3.5-4.5 kg. The animal models described herein relied on the three most common clinical causes for CP, such as guidewire-induced, balloon catheter bursting, and device oversizing. Moreover, the sealing capacity of covered stent grafts was assessed for each of these models by means of angiography. RESULTS: We herein report a rabbit iliac artery perforation model using three different types of vessel perforation that closely mimic the clinical setting, such as guidewire-induced, balloon catheter rupture, and device oversizing. Using the same rabbit iliac perforation model, we additionally assessed the sealing capacity of a covered stent graft for each model. CONCLUSIONS: The novel rabbit iliac artery perforation models, as described in this report, represent promising animal testing approaches. While their setting is very similar to the real-life context encountered in humans, all three models are based on an animal model that is ideally suited for evaluating the sealing capacity and performance of new medical devices for humans.

Prevalence and Mortality of Venous Leg Diseases of the Deep Veins: An Observational Cohort Study Based on German Health Claims Data.

This study estimates the prevalence and mortality of diseases of the deep veins of the legs such as deep vein thrombosis (DVT), postthrombotic syndrome (PTS), and venous leg ulceration (VLU). We used a random sample of 250 000 patients at age 50+ years of the register of the Allgemeine Ortskrankenkasse from 2004 to 2015. Selected manifestations of venous diseases assumed as risk factors for mortality were analyzed using Cox models while adjusting for various basic demographic and health characteristics. The prevalence in 2004 was 0.05% for DVT of the femoral veins, 0.50% for DVT of any deep veins, 0.86% for PTS, and 0.91% for VLU. The mortality rate in 2004 to 2015 was 20.40 deaths/100 person-years for DVT of the femoral veins, 10.69 for DVT of any deep veins, 4.34 for PTS, and 7.02 for VLU. The model revealed a 35% higher risk (p < .001) in patients with any DVT, an 88% higher mortality (p < .001) for femoral DVT, a 23% higher risk (p < .001) for VLU, and no health disadvantage in persons with PTS. Our study revealed an increased mortality for patients with VLU and DVT. Even after adjustment for embolic events and infections of the venous ulcers mortality remained significantly higher.

Can Recognition of Spinal Ischemia Be Improved? Application of Motor-Evoked Potentials, Serum Markers, and Breath Gas Analysis in an Acutely Instrumented Pig Model.

BACKGROUND: Paraplegia due to spinal cord ischemia (SCI) is a serious complication after repair of thoracoabdominal aortic aneurysms. For prevention and early treatment of spinal ischemia, intraoperative monitoring of spinal cord integrity is essential. This study was intended to improve recognition of SCI through a combination of transcranial motor-evoked potentials (tc-MEPs), serum markers, and innovative breath analysis. METHODS: In 9 female German Landrace pigs, tc-MEPs were captured, markers of neuronal damage were determined in blood, and volatile organic compounds (VOCs) were analyzed in exhaled air. After thoraco-phrenico-laparotomy, SCI was initiated through sequential clamping (n = 4) or permanently ligating (n = 5) SAs of the abdominal and thoracic aorta in caudocranial orientation until a drop in the tc-MEPs to at least 25% of the baseline was recorded. VOCs in breath were determined by means of solid-phase microextraction coupled with gas chromatography-mass spectrometry. After waking up, clinical and neurological status was evaluated (Tarlov score). Spinal cord histology was obtained in postmortem. RESULTS: Permanent vessel ligature induced a worse neurological outcome and a higher number of necrotic motor neurons compared to clamping. Changes of serum markers remained unspecific. After laparotomy, exhaled acetone and isopropanol showed highest concentrations, and pentane and hexane increased during ischemia-reperfusion injury. CONCLUSIONS: To mimic spinal ischemia occurring in humans during aortic aneurysm repair, animal models have to be meticulously evaluated concerning vascular anatomy and function. Volatiles from breath indicated metabolic stress during surgery and oxidative damage through ischemia reperfusion. Breath VOCs may provide complimentary information to conventional monitoring methods.

In Vivo Testing of Extracorporeal Membrane Ventilators: iLA-Activve Versus Prototype I-Lung.

A side-by-side comparison of the decarboxylation efficacy of two pump-driven venovenous extracorporeal lung assist devices, i.e., a first prototype of the new miniaturized ambulatory extracorporeal membrane ventilator, I-lung versus the commercial system iLA-activve for more than a period of 72 hours in a large animal model. Fifteen German Landrace pigs were anesthetized and underwent mechanical hypoventilation to induce severe hypercapnia. Decarboxylation was accomplished by either the I-lung or the iLA-activve via a double lumen catheter in the jugular vein. Sham-operated pigs were not connected to extracorporeal devices. Cardiovascular, respiratory, and metabolic parameters were continuously monitored, combined with periodic arterial blood sampling for subsequent clinical blood diagnostics, such as gas exchange, hemolysis, coagulation parameters, and cytokine profiles. At the termination of the studies, lung tissue was harvested and examined histologically for pulmonary morphology and leukocyte tissue infiltration. Both extracorporeal devices showed high and comparable efficacy with respect to carbon dioxide elimination for more than 72 hours and were not associated with either bleeding events or clotting disorders. Pigs of both groups showed cardiovascular and hemodynamic stability without marked differences to sham-operated animals. Groups also did not differ in terms of inflammatory and metabolic parameters. We established a preclinical in vivo porcine model for comparative long-term testing of I-lung and iLA-activve. The I-lung prototype proved to be safe and feasible, providing adequate decarboxylation without any adverse events. Once translated into the clinical treatment, the new miniaturized and transportable I-lung device might represent a promising tool for treating awake and mobilized patients with decompensated pulmonary disorders.

Development of a sirolimus-eluting poly (L-lactide)/poly(4-hydroxybutyrate) absorbable stent for peripheral vascular intervention.

Fully absorbable drug-eluting stent platforms are currently entering the clinical arena for the interventional treatment of coronary artery disease. This new technology also holds potential for application in peripheral vascular settings. Our study reports on the development of a sirolimus- (SIR) eluting absorbable polymer stent made from a blend of poly(l-lactide) and poly(4-hydroxybutyrate) (PLLA/P4HB) for peripheral vascular intervention. Stent prototypes were laser-cut from PLLA/P4HB tubes (I.D.=2.2 mm, t=250 µm), spray-coated with different PLLA/P4HB/SIR solutions, and bench-tested to determine expansion properties, fatigue, trackability and in vitro drug release kinetics. The stent prototypes were expanded with a 5.0 × 20 mm balloon catheter, and exhibited a recoil of 3.6% upon balloon deflation. Stent collapse pressure of 0.4 bar (300 mm Hg) was measured under external pressure load. Sustained scaffolding properties were observed in vitro over 14 weeks of radial fatigue loading (50 ± 25 mm Hg at 1.2 Hz). Trackability was demonstrated in bench tests with an 8 French contralateral introducer sheath. SIR release kinetics were adjusted over a broad range by varying the PLLA/P4HB ratio of the coating matrix. The newly developed absorbable SIR-eluting PLLA/P4HB stent successfully fulfilled the requirements for peripheral vascular intervention under in vitro conditions.

Volatile breath biomarkers for patient monitoring during haemodialysis.

Patients with end-stage renal disease (ESRD) are at risk for a numerous complications. This study was intended to evaluate breath analysis for monitoring and therapy initiation under haemodialysis (HD). Exhaled alveolar air from 30 ESRD patients during 4 h thrice-weekly HD was analysed by means of HS-SPME-GC-MS. Venous blood samples were taken for determination of conventional serum parameters. Exhaled concentrations of isoprene (10-589 ppbV) were dropped at initiation of HD and increased at the end of HD. Isoprene concentration changes were similar to changes of serum LDH activities. Variation of exhaled acetone concentrations (59 to 8509 ppbV) was significantly lower in diabetic patients when compared to non-diabetics. Exhaled pentane (0.3 to 12 ppbV) increased at onset of HD and returned to baseline levels afterwards. Benzene concentrations showed typical washout characteristics. Ethanol and DMS concentrations remained constant during HD. Breath analysis can be used to recognize oxidative stress, metabolic conditions and haemolysis during HD. Hence, non-invasive breath testing could be used to monitor ESRD patients under HD and prevent them from being affected by well-known detrimental side effects of renal replacement therapy.

Breath analysis during one-lung ventilation in cancer patients.

Noninvasive breath analysis may provide valuable information for cancer recognition if disease-specific volatile biomarkers could be identified. In order to compare nondiseased and diseased tissue in vivo, this study took advantage of the special circumstances of one-lung ventilation (OLV) during lung-surgery. 15 cancer patients undergoing lung resection with OLV were enrolled. From each patient, alveolar breath samples were taken separately from healthy and diseased lungs before and after tumour resection. Volatile substances were pre-concentrated by means of solid-phase microextraction, and were separated, identified and quantified by means of gas chromatography-mass spectrometry. Different classes of volatile substances could be identified according to their concentration profiles. Due to prolonged fasting and activation of lipolysis, concentrations of endogenous acetone significantly increased during surgery. Exogenous substances, such as benzene or cyclohexanone, showed typical washout exhalation kinetics. Exhaled concentrations of potentially tumour associated substances, such as butane or pentane, were different for nondiseased and diseased lungs and decreased significantly after surgery. Separate analysis of volatile substances exhaled from healthy and diseased lungs in the same patient, together with thorough consideration of substance origins and exhalation kinetics offers unique opportunities of biomarker recognition and evaluation.

Needle trap micro-extraction for VOC analysis: effects of packing materials and desorption parameters.

Combining advantages of SPE and SPME needle trap devices (NTD) represent promising new tools for a robust and reproducible sample preparation. This study was intended to investigate the effect of different packing materials on efficacy and reproducibility of VOC analysis by means of needle trap micro extraction (NTME). NTDs with a side hole design and containing different combinations of PDMS, DVB and Carbopack X and Carboxen 1000 and NTDs containing a single layer organic polymer of methacrylic acid and ethylene glycol dimethacrylate were investigated with respect to reproducibility, LODs and LOQs, carry over and storage. NTDs were loaded with VOC standard gas mixtures containing saturated and unsaturated hydrocarbons, oxygenated and aromatic compounds. Volatile substances were thermally desorbed from the NTDs using fast expansive flow technique and separated, identified and quantified by means of GC-MS. Optimal desorption temperatures between 200 and 290°C could be identified for the different types of NTDs with respect to desorption efficiency and variation. Carry over was below 6% for polymer packed needles and up to 67% in PDMS/Carboxen 1000 NTDs. Intra and inter needle variation was best for polymer NTDs and consistently below 9% for this type of NTD. LODs and LOQs were in the range of some ng/L. Sensitivity of the method could be improved by increasing sample volume. NTDs packed with a copolymer of methacrylic acid and ethylene glycol dimethacrylate were universally applicable for sample preparation in VOC analysis. If aromatic compounds were to be determined DVB/Carboxen 1000 and DVB/Carbopack X/Carboxen 1000 devices could be considered as an alternative. PDMS/Carbopack X/Carboxen 1000 NTDs may represent a good alternative for the analysis of hydrocarbons and aldehydes. NTME represents a powerful tool for different application areas, from environmental monitoring to breath analysis.

Drug detection in breath: effects of pulmonary blood flow and cardiac output on propofol exhalation.

Breath analysis could offer a non-invasive means of intravenous drug monitoring if robust correlations between drug concentrations in breath and blood can be established. In this study, propofol blood and breath concentrations were determined in an animal model under varying physiological conditions. Propofol concentrations in breath were determined by means of two independently calibrated analytical methods: continuous, real-time proton transfer reaction mass spectrometry (PTR-MS) and discontinuous solid-phase micro-extraction coupled with gas chromatography mass spectrometry (SPME-GC-MS). Blood concentrations were determined by means of SPME-GC-MS. Effects of changes in pulmonary blood flow resulting in a decreased cardiac output (CO) and effects of dobutamine administration resulting in an increased CO on propofol breath concentrations and on the correlation between propofol blood and breath concentrations were investigated in seven acutely instrumented pigs. Discontinuous propofol determination in breath by means of alveolar sampling and SPME-GC-MS showed good agreement (R(2)=0.959) with continuous alveolar real-time measurement by means of PTR-MS. In all investigated animals, increasing cardiac output led to a deterioration of the relationship between breath and blood propofol concentrations (R(2)=0.783 for gas chromatography-mass spectrometry and R(2)=0.795 for PTR-MS). Decreasing pulmonary blood flow and cardiac output through banding of the pulmonary artery did not significantly affect the relationship between propofol breath and blood concentrations (R(2)>0.90). Estimation of propofol blood concentrations from exhaled alveolar concentrations seems possible by means of different analytical methods even when cardiac output is decreased. Increases in cardiac output preclude prediction of blood propofol concentration from exhaled concentrations.

Phase-resolved real-time breath analysis during exercise by means of smart processing of PTR-MS data.

Separation of inspiratory, mixed expired and alveolar air is indispensable for reliable analysis of VOC breath biomarkers. Time resolution of direct mass spectrometers often is not sufficient to reliably resolve the phases of a breathing cycle. To realise fast on-line breath monitoring by means of direct MS utilising low-fragmentation soft ionisation, a data processing algorithm was developed to identify inspiratory and alveolar phases from MS data without any additional equipment. To test the algorithm selected breath biomarkers (acetone, isoprene, acetaldehyde and hexanal) were determined by means of quadrupole proton transfer reaction mass spectrometry (PTR-MS) in seven healthy volunteers during exercise on a stationary bicycle. The results were compared to an off-line reference method consisting of controlled alveolar breath sampling in Tedlar® bags, preconcentration by solid-phase micro extraction (SPME), separation and identification by GC-MS. Based on the data processing method, quantitative attribution of biomarkers to inspiratory, alveolar and mixed expiratory phases was possible at any time during the experiment, even under respiratory rates up to 60/min. Alveolar concentrations of the breath markers, measured by PTR-MS ranged from 130 to 2,600 ppb (acetone), 10 to 540 ppb (isoprene), 2 to 31 ppb (acetaldehyde), whereas the concentrations of hexanal were always below the limit of detection (LOD) of 3 ppb. There was good correlation between on-line PTR-MS and SPME-GC-MS measurements during phases with stable physiological parameters but results diverged during rapid changes of heart rate and minute ventilation. This clearly demonstrates the benefits of breath-resolved MS for fast on-line monitoring of exhaled VOCs.

Breath biomarkers for lung cancer detection and assessment of smoking related effects--confounding variables, influence of normalization and statistical algorithms.

BACKGROUND: Up to now, none of the breath biomarkers or marker sets proposed for cancer recognition has reached clinical relevance. Possible reasons are the lack of standardized methods of sampling, analysis and data processing and effects of environmental contaminants. METHODS: Concentration profiles of endogenous and exogenous breath markers were determined in exhaled breath of 31 lung cancer patients, 31 smokers and 31 healthy controls by means of SPME-GC-MS. Different correcting and normalization algorithms and a principal component analysis were applied to the data. RESULTS: Differences of exhalation profiles in cancer and non-cancer patients did not persist if physiology and confounding variables were taken into account. Smoking history, inspired substance concentrations, age and gender were recognized as the most important confounding variables. Normalization onto PCO2 or BSA or correction for inspired concentrations only partially solved the problem. In contrast, previous smoking behaviour could be recognized unequivocally. CONCLUSION: Exhaled substance concentrations may depend on a variety of parameters other than the disease under investigation. Normalization and correcting parameters have to be chosen with care as compensating effects may be different from one substance to the other. Only well-founded biomarker identification, normalization and data processing will provide clinically relevant information from breath analysis.

Automated needle trap heart-cut GC/MS and needle trap comprehensive two-dimensional GC/TOF-MS for breath gas analysis in the clinical environment.

This study was intended to evaluate low-volume (20 mL) multibed needle trap (NTD) sampling combined with heart-cut gas chromatography/mass spectrometry (GC/MS) and comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOF-MS) for trace gas analysis under clinical conditions. NTDs, high-throughput automatic desorption and separation systems, were tested in vitro and within a study in 11 patients undergoing cardiac surgery with respect to reproducibility, reliability, and clinical applicability. NTD-heart-cut GC/MS analysis of standard mixtures containing different volatile organic compounds (VOCs) yielded relative standard deviations (RSDs) from 4.0% to 18.5%. Substance adsorption was stable for 1 day if NTDs were closed on both ends and was stable for approximately 7.8 h when NTD tip ends had to be left open during autosampler storage. Even in the presence of high concentrations of contaminants linearity of heart-cut GC/MS was conserved. In patients' breath potential biomarkers could be determined even in the presence of very high concentrations of sevoflurane. Profiles of blood-borne biomarkers, intravenous drugs, and clinical contaminants were characterized. Comprehensive GC x GC/TOF-MS may be used as a screening tool for new biomarkers, if patterns are generated from deconvoluted normalized areas. Needle trap sampling in combination with hyphenated chromatographic techniques can thus be used to provide well-tailored solutions for complex problems occurring in clinical breath analysis.

Multibed needle trap devices for on site sampling and preconcentration of volatile breath biomarkers.

To facilitate their use in trace gas analysis, the adsorption capacity of needle trap devices (NTDs) was increased by combining three adsorbent materials and increasing total adsorbent amount. The use of 22 gauge needles, application of internally expanding desorptive flow technique without cryofocusation and a new on site alveolar sampling method for NTDs provided sensitivity in the parts per trillion range of VOC concentrations without loosing precision or linearity. LODs were 0.4 ng/L for isoprene, 0.5 ng/L for dimethyl sulphide, 0.9 ng/L for 2-butenal, 1.0 ng/L for hexane, 1.2 ng/L for pentane, 2.3 ng/L for hexanal, 5.3 ng/L for pentanal, and 8.3 ng/L for acetone. R of calibration curves were consistently >0.98. Loss of volatile aldehydes during storage for 7 days was less than 10%. Needle trap devices packed with more than one adsorbent material represent a promising alternative to SPE and SPME for analysis of volatile organic compounds in the low parts per billion/parts per trillion range. Crucial problems of clinical breath analysis concerning sensitivity of analytical methods, limited stability, and decomposition of breath compounds during sampling and storage could be solved.

Impact of sampling procedures on the results of breath analysis.

The impact of different sampling techniques on the results of breath analysis was to be assessed in this study. Alveolar, mixed expiratory and time-controlled samples were collected from ten volunteers and from eight lung cancer patients. Breath sampling was visually controlled by means of capnometry. PCO(2) and 13 VOCs were determined. Mixed expiratory sampling yielded 25% lower concentrations of CO(2) and blood-borne VOCs. Time-controlled sampling generated high variation of results. Ratios C(alv)/C(mixed) were >1.5 for CO(2), acetone and isoprene, and <1 for isopropanol, 2-butanone and hexanal. Acetonitrile, butane, dimethylsulfide, pentane, butanal, benzene and hexane showed 1.5 > C(alv)/C(mixed) > 1. The ratio C(alv)/C(mixed) of CO(2), acetone and isoprene was different in healthy volunteers and lung cancer patients. Alveolar samples showed the highest concentrations of endogenous and lowest concentration of exogenous substances. Sampling can impact results in breath analysis. Valuable information can be obtained from ratios of alveolar and mixed expired concentrations.

Monitoring of oxidative and metabolic stress during cardiac surgery by means of breath biomarkers: an observational study.

BACKGROUND: Volatile breath biomarkers provide a non-invasive window to observe physiological and pathological processes in the body. This study was intended to assess the impact of heart surgery with extracorporeal circulation (ECC) onto breath biomarker profiles. Special attention was attributed to oxidative or metabolic stress during surgery and extracorporeal circulation, which can cause organ damage and poor outcome. METHODS: 24 patients undergoing cardiac surgery with extracorporeal circulation were enrolled into this observational study. Alveolar breath samples (10 mL) were taken after induction of anesthesia, after sternotomy, 5 min after end of ECC, and 30, 60, 90, 120 and 150 min after end of surgery. Alveolar gas samples were withdrawn from the circuit under visual control of expired CO2. Inspiratory samples were taken near the ventilator inlet. Volatile substances in breath were preconcentrated by means of solid phase micro extraction, separated by gas chromatography, detected and identified by mass spectrometry. RESULTS: Mean exhaled concentrations of acetone, pentane and isoprene determined in this study were in accordance with results from the literature. Exhaled substance concentrations showed considerable inter-individual variation, and inspired pentane concentrations sometimes had the same order of magnitude than expired values. This is the reason why, concentrations were normalized by the values measured 120 min after surgery. Exhaled acetone concentrations increased slightly after sternotomy and markedly after end of ECC. Exhaled acetone concentrations exhibited positive correlation to serum C-reactive protein concentrations and to serum troponine-T concentrations. Exhaled pentane concentrations increased markedly after sternotomy and dropped below initial values after ECC. Breath pentane concentrations showed correlations with serum creatinine (CK) levels. Patients with an elevated CK-MB (myocardial&brain)/CK ratio had also high concentrations of pentane in exhaled air. Exhaled isoprene concentrations raised significantly after sternotomy and decreased to initial levels at 30 min after end of ECC. Exhaled isoprene concentrations showed a correlation with cardiac output. CONCLUSION: Oxidative and metabolic stress during cardiac surgery could be assessed continuously and non-invasively by means of breath analysis. Correlations between breath acetone profiles and clinical conditions underline the potential of breath biomarker monitoring for diagnostics and timely initiation of life saving therapy.