Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans.

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.

Whole body UVA irradiation lowers systemic blood pressure by release of nitric oxide from intracutaneous photolabile nitric oxide derivates.

RATIONALE: Human skin contains photolabile nitric oxide derivates like nitrite and S-nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. OBJECTIVE: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. METHODS AND RESULTS: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm(2)) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S-nitrosothiols 2.3-fold, and the subfraction of dermal S-nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled (15)N-nitrite, whole body UVA irradiation (20 J/cm(2)) induced significant levels of (15)N-labeled S-nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11+/-2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated (R(2)=0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26+/-7%), with increased flow mediated vasodilation of the brachial artery (+68+/-22%), and with decreased forearm vascular resistance (-28+/-7%). CONCLUSIONS: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.

Intercomparison of infrared cavity leak-out spectroscopy and gas chromatography-flame ionization for trace analysis of ethane.

Comparison of two different methods for the measurement of ethane at the parts-per-billion (ppb) level is reported. We used cavity leak-out spectroscopy (CALOS) in the 3 microm wavelength region and gas chromatography-flame ionization detection (GC-FID) for the analysis of various gas samples containing ethane fractions in synthetic air. Intraday and interday reproducibilities were studied. Intercomparing the results of two series involving seven samples with ethane mixing ratios ranging from 0.5 to 100 ppb, we found a reasonable agreement between both methods. The scatter plot of GC-FID data versus CALOS data yields a linear regression slope of 1.07 +/- 0.03. Furthermore, some of the ethane mixtures were checked over the course of 1 year, which proved the long-term stability of the ethane mixing ratio. We conclude that CALOS shows equivalent ethane analysis precision compared to GC-FID, with the significant advantage of a much higher time resolution (<1 s) since there is no requirement for sample preconcentration. This opens new analytical possibilities, e.g., for real-time monitoring of ethane traces in exhaled human breath.

NO detection in biological samples: differentiation of 14 NO and 15 NO using infrared laser spectroscopy.

Accurate characterization of the biochemical pathways of nitric oxide (NO) is essential for investigations in the field of NO research. To analyze the different reaction pathways of enzymatic and non-enzymatic NO formation, determination of the source of NO is crucial. Measuring NO-related products in biological samples distinguishing between (14)NO and (15)NO offers the opportunity to specifically analyze NO signaling in blood and tissue. The aim of this study was to establish a highly sensitive technique for the specific measurement of NO in an isotopologue-selective manner in biological samples. With the cavity leak-out spectroscopy setup (CALOS) a differentiation between (14)NO and (15)NO is feasible. We describe here the employment of this method for measurements in biological samples. Certified gas mixtures of (14)NO/N(2) and (15)NO/N(2) were used to calibrate the system. (14)NO2- and (15)NO2- of aqueous and biological samples were reduced in a triiodide solution, and the NO released was detected via CALOS. Gas-phase chemiluminescence detection (CLD) was used for evaluation. The correlation received for both methods for the detection of NO in the gas phase was r=0.999, p<0.0001. Results obtained using aqueous and biological samples verified that CALOS enables NO measurements with high accuracy (detection limit for (14)NO2- 0.3 pmol and (15)NO2- 0.5 pmol; correlation (14)NO: p<0.0001, r=0.975, (15)NO: p<0.0001, r=0.969). The CALOS assay represents an extension of NO measurements in biological samples, allowing specific investigations of enzymatic and non-enzymatic NO formation and metabolism in a variety of samples.

Is exhaled carbon monoxide level associated with blood glucose level? A comparison of two breath analyzing methods.

The level of exhaled carbon monoxide (eCO) is considered a marker of oxidative stress in diabetes. Previous findings indicated that eCO levels correlated with blood glucose level. The aim of this work was to apply and compare two independent analyzing methods for eCO after oral glucose administration. Glycemia, eCO, and exhaled hydrogen were measured before and after oral administration of glucose. Six healthy nonsmoking volunteers participated. For eCO analysis, we used two methods: a commercially available electrochemical sensor, and a high-precision laser spectrometer developed in our laboratory. The precision of laser-spectroscopic eCO measurements was two orders of magnitude better than the precision of the electrochemical eCO measurement. eCO levels measured by laser spectrometry after glucose administration showed a decrease of 4.1%+/-1.5% compared to the baseline (p<0.05). Changes in the eCO measured by the electrochemical sensor were not significant (p=0.08). Exhaled hydrogen levels increased by 40% within the first 10 min after glucose administration (p<0.05). The previous finding that the glycemia increase after glucose administration was associated with a significant increase in eCO concentrations was not confirmed. We propose that previous eCO measurements with electrochemical sensors may have been affected by cross sensitivity to hydrogen.

Ultra sensitive trace gas detection for biomedical applications.

We present an overview of our recent progress on spectroscopic trace gas detection for biomedical applications. The latest developments of cavity-enhanced spectroscopy as well as magnetic rotation spectroscopy lead to unprecedented sensitivity and specificity. The current detection limits of our laser spectroscopic approaches are in the picomolar to nanomolar range, depending on the molecular compound. The time resolution of the measurements is down to the sub-second range. This very high sensitivity and time resolution open up exciting perspectives for novel analytical tasks in biomedical research and clinical diagnosis.

Enzyme-independent nitric oxide formation during UVA challenge of human skin: characterization, molecular sources, and mechanisms.

Many of the local UV-induced responses including erythema and edema formation, inflammation, premature aging, and immune suppression can be influenced by nitric oxide synthase (NOS)-produced NO which is known to play a pivotal role in cutaneous physiology. Besides NOS-mediated NO production, UV radiation might trigger an enzyme-independent NO formation in human skin by a mechanism comprising the decomposition of photo-reactive nitrogen oxides. Therefore, we have examined the chemical-storage forms of potential NO-generating agents, the mechanisms and kinetics of their decomposition, and their biological relevance. In normal human skin specimens we find nitrite and S-nitrosothiols (RSNO) at concentrations 25- or 360-fold higher than those found in plasma of healthy volunteers. UVA irradiation of human skin leads to high-output formation of bioactive NO due to photo-decomposition of RSNO and nitrite which represents the primary basis for NO formation during UVA exposure. Interestingly, reduced thiols strongly augment photo-decomposition of nitrite and are essential for maximal NO release. The enzyme-independent NO formation found in human skin opens a completely new field in cutaneous physiology and will extend our understanding of mechanisms contributing to skin aging, inflammation, and cancerogenesis.

The presence of nitrite during UVA irradiation protects from apoptosis.

Nitrite occurs ubiquitously in biological fluids such as blood and sweat, representing an oxidation product of nitric oxide. Nitrite has been associated with a variety of adverse effects such as mutagenicity, carcinogenesis, and toxicity. In contrast, here we demonstrate that the presence of nitrite, but not nitrate, during irradiation of endothelial cells in culture exerts a potent and concentration-dependent protection against UVA-induced apoptotic cell death. Protection is half-maximal at a concentration of 3 mM, and complete rescue is observed at 10 mM. Nitrite-mediated protection is mediated via inhibition of lipid peroxidation in a similar manner as seen with butylated hydroxytoluene, a known inhibitor of lipid peroxidation. Interestingly, nitrite-mediated protection is completely abolished by coincubation with the NO scavenger cPTIO. Using electron paramagnetic resonance (EPR) spectroscopy or Faraday modulation spectroscopy, we directly prove UVA-induced NO formation in solutions containing nitrite. In conclusion, evidence is presented that nitrite represents a protective agent against UVA-induced apoptosis due to photodecomposition of nitrite and subsequent formation of NO.

Time resolved simultaneous detection of 14NO and 15NO via mid-infrared cavity leak-out spectroscopy.

We present a ring-down absorption spectrometer based on a continuous-wave CO laser in the mid-infrared spectral region near lambda = 5 microm. Using a linear ring-down cavity (length: 0.5 m) with high reflective mirrors (R = 99.988 %), we observed a noise-equivalent absorption coefficient of 3 x 10(-10) cm(-1)Hz(-1/2). This corresponds to a noise-equivalent concentration of 800 parts per trillion (ppt) for (14)NO and 40 ppt for (15)NO in 1 s averaging time. We achieve a time resolution of 1 s which allows time resolved simultaneous detection of the two N isotopes. The delta(15)N value was obtained with a precision of +/-1.2 per thousand in a sample with a NO fraction of 11 ppm. The simultaneous detection enables the use of (15)NO as a tracer molecule for endogenous biomedical processes.

Online recording of ethane traces in human breath via infrared laser spectroscopy.

A method is described for rapidly measuring the ethane concentration in exhaled human breath. Ethane is considered a volatile marker for lipid peroxidation. The breath samples are analyzed in real time during single exhalations by means of infrared cavity leak-out spectroscopy. This is an ultrasensitive laser-based method for the analysis of trace gases on the sub-parts per billion level. We demonstrate that this technique is capable of online quantifying of ethane traces in exhaled human breath down to 500 parts per trillion with a time resolution of better than 800 ms. This study includes what we believe to be the first measured expirograms for trace fractions of ethane. The expirograms were recorded after a controlled inhalation exposure to 1 part per million of ethane. The normalized slope of the alveolar plateau was determined, which shows a linear increase over the first breathing cycles and ends in a mean value between 0.21 and 0.39 liter-1. The washout process was observed for a time period of 30 min and was modelled by a threefold exponential decay function, with decay times ranging from 12 to 24, 341 to 481, and 370 to 1770 s. Our analyzer provides a promising noninvasive tool for online monitoring of the oxidative stress status.

Mid-infrared laser spectroscopy for online analysis of exhaled CO.

Carbon monoxide (CO) detection in human breath is the focus of much research because of CO's possible use as a marker molecule for different diseases. Detecting CO in human breath remains a tough challenge because of the low concentrations of CO (ppm range) that must be detected. Another problem is that many other molecules, which can be found in human breath, can interfere in these measurements. Additionally, a time resolution of less than 1 s is needed to resolve the CO curve of an exhalation. In this study (13)CO instead of (12)CO concentration is measured. The measurements are performed with a cavity leak-out spectroscopy system. The system's properties match the above-mentioned specifications for measurements, even of the rare isotopologue with high specificity, a time resolution of less than 1 s and a detection limit of 7 ppb Hz(-1/2). Two investigations are presented here. The first is a measurement showing intraday changes between 9.5% and 23.3% of the exhaled CO level due to vigorous exercise. The second shows a long-term observation of the CO base level revealing natural variations in the recorded CO concentration. The covered time period is 25 weeks during which the differences between the minimum and maximum CO levels for each test case reached 84%.

Infrared laser spectroscopy for online recording of exhaled carbon monoxide-a progress report.

Despite the growing number of reports on breath CO measurements, the development of rapid and sensitive analysis techniques for measurements of this breath constituent still remains a challenge. We demonstrate the application of infrared laser spectroscopy for exhaled CO analysis. The breath samples are analyzed in real-time during single exhalations by means of cavity ring-down spectroscopy. This is an ultra-sensitive laser-based method for the analysis of trace gases with precision on the ppb level (parts per billion). The noise-equivalent CO level of this method is 7 ppb Hz(-1/2); the time resolution is around 1 s. The expirograms were recorded with exhalation flow rates varying from 4 l min(-1) up to 50 l min(-1). Alveolar phase (phase III) of expiration shows a remarkable flow-rate dependence. Also, expirograms were recorded after a breath holding time between 0 s and 60 s. The normalized slope of the alveolar plateau (S(n)) was determined, which is between 0.004 l(-1) and 0.15 l(-1).

Parts per trillion sensitivity for ethane in air with an optical parametric oscillator cavity leak-out spectrometer.

Spectroscopic detection of ethane in the 3-microm wavelength region was performed by means of a cw optical parametric oscillator and cavity leak-out. We achieved a minimum detectable absorption coefficient of 1.6 x 10(-10) cm 1/square root of Hz, corresponding to an ethane detection limit of 6 parts per trillion/square root of Hz. For 3-min integration time the detection limit was 0.5 parts per trillion. The levels are to our knowledge the best demonstrated so far. These frequency-tuning capabilities facilitated multigas analysis with simultaneous monitoring of ethane, methane, and water vapor in human breath.