A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

The overall goal of this procedure is to measure the amount of free radicals in mainstream tobacco smoke and to assess the scavenging capacity of several antioxidants. This is accomplished by first preparing filters containing several different scavenger compounds as well as controls without scavenger filter. Preparation is followed by smoking simulation with the smoke of research cigarettes, passing through a spin trapping solution.

Electron spin resonance, abbreviated ESR is then used to record spectra of trapped radicals as well as of reference samples. Next obtained ESR Spectra are subjected to routine analysis in order to separate the signal of the trapped gas phase radicals from the background and to quantify the radical concentration using a reference sample. Ultimately, results show quantitative information on the content of free radicals in mainstream cigarette smoke and estimates of scavenging capacity of natural antioxidants through spin trapping ESR spectroscopy.

The main advantage of this technique over the existing methods of tobacco smoke analysis like gas chromatography, is that ESR is specifically sensitive to to free radicals. The implications of this technique can help in reducing hazardous effects of tobacco smoke because theoreticals is a major group of carcinogen. This method can provide insight in the studying free radical in the gasket smoke, and it can also be applied to other systems such as industrial smoke exhaust and smacked The National Biomedical Research Center for electron spin resonance technology, also known as a cert, is an outgrowth of our extensive experience in developing modern ESR technology.

For researchers engaged in electrons spin resonance, we offer a wide variety of service for them and for the biomedical community. We make our facilities regularly available To introduce antioxidants into the cigarette filter. First, dissolve the antioxidants, pycnogenol, and grapee extract in 95%ethanol and dissolve lycopene and acetone.

Adjust the solvent volumes depending on the antioxidants solubility. Next, combine the dissolved antioxidants with activated carbon by stirring in anaerobic conditions for approximately 12 hours. Anaerobic conditions are achieved by bubbling Argonne through the mixture while stirring.

Following incubation, filter the solution and dry under a vacuum. Then introduce the antioxidants and carbon into a conventional acetate filter by first cutting the filter into two pieces. Insert the coated plant antioxidants between two pieces of the filter to form a filter antioxidant filter sandwich, and reinsert it into the cigarettes paper cylinder.

Prepare the control filter in the same way as the antioxidant filter, except not to add the antioxidant prior to experimental setup. The research cigarettes should be unpacked and kept in a constant humidity environment using a saturated sodium bromide solution for a minimum of two days. Retrieve the cigarette rods containing tobacco and attach to the combined filter.

Perform smoking simulation for routine analysis at ambient temperature using a single port smoking device, which consists of a water aspirator or vacuum pump slash air compressor connected to the spin trapping assembly through a T junction with one end open. Use a flow meter to check the gas flow and set at approximately 2.2 standard cubic feet per hour by adjusting a valve placed between the pump and the vent. Smoke the research cigarettes using an intense puff volume of 35 milliliters for a duration of two seconds and repeat every 60 seconds.

The open end of the smoking device should be plugged to perform puffs and left open between puffs. While the end is unplugged, there is little draft through the cigarette, which corresponds to the conditions between puffs. Take 10 intense puffs for each cigarette collecting gas phase free radicals by passing the mainstream cigarette smoke through a Cambridge filter pad, and then introducing into the spin trapping solution.

The liquid nitrogen trap removes water vapor from the mainstream smoke, but does not substantially affect the gas phase radicals. After the last puff transfer an aliquot of the bubbled trapping solution into an approximately 10 inch long, three millimeter internal diameter glass sample tube. Since oxygen broadens ESR spectra and decreases the method sensitivity, deoxy oxygenate the solution using the freeze pump thaw method by first dipping the tube into liquid nitrogen, then evacuating the solution with an oil plump, and then thawing the solution under Argonne.

Repeat this freeze pump thaw method several times. Next, seal the tube under vacuum. The tube is now ready.

For further ESR measurements, record X-band ESR spectra on a Bruker EMX spectrometer at a frequency of 9.34 gigahertz. Under standard conditions, consult the written protocol for typical spectrometer settings used. For these experiments perform 25 ESR accumulations, which typically take approximately 20 minutes.

Although the spin trap attics are relatively stable at the experimental conditions after 12 hours, the intensity of the ESR signals in benzene solution decreased by a factor of five. To quantify the concentration of the trapping addict, integrate the initial first derivative ESR spectrum. The resulting absorption spectrum shows a broad singlet background, most likely due to soot and tar products contained in the smoke.

After subtraction of this background, integrate the separated triplet of the trapping addict. One more time to get the free radical concentration. The values of double integrals for the spin trapping solution are compared with those for a solution of a nitrox radical tempo with known concentration from the experiment demonstrated here, as well as additional calculations found in the written protocol.

A free radical concentration in the gas phase of mainstream cigarette smoke of approximately 7.1 times 10 to the minus nine molar was obtained. From this, it was determined that the total number of radicals trapped from the gas phase of a whole cigarette is around 1.5 times 10 to the 15th. During this experiment, initial measurements led to weak ESR signals and low reproducibility due to the moisture in the smoke flow.

To resolve this problem, a liquid nitrogen trap was added to the smoking simulation device between the Cambridge filter and the spin trap solution. The liquid nitrogen trap removed water from the mainstream cigarette smoke flow by quick freezing and capturing it on the inner wall of the glass tube. This greatly improved ESR signals and allowed for highly reproducible results.

Different levels of the scavenging effect of plant antioxidants on gas phase, free radicals in mainstream cigarette smoke were observed and their scavenging rates are presented. Lycopene and grape seed extract showed the highest rates while lower rate was observed for pycnogenol. Once master, this technique can be done in less than one hour if performed properly.

Once attempting the procedure, it's important to pass the smoke through a liquid nitrogen trap. It improves the quality of a SR spectra and the reproducibility once developed. The technique helped the researchers in health and environment to explore and to elevate the presence of health threatening free radicals in various volatile compounds.

After watching the video, you should have a good understanding of how to quantify free radicals in the mainstream tobacco smoke in order to reduce hazardous effects of smoking.