[MUSIC] In this lecture on chemicals in health, we will discuss two studies that took place in New York State that investigate exposure to environmental tobacco smoke. Also known as secondhand smoke. My name is Kenneth Aldous, and I’m the director of the Division of Environmental Health Sciences at the Wadsworth Center New York State Department of Health. In this slide chart I will discuss the ways that exposure to tobacco smoke can be measured in using the chemical cotinine as a biomarker in our blood and saliva. Collection of samples to monitor our exposure. The laboratory method used to determine trace levels of cotinine in these body fluids. And the results of two studies in which cotinine was measured to assess the impact of smoking ban legislation. In New York state. Cigarettes, cigars, and pipe tobacco are made from dried tobacco leaves and ingredients are added for flavor, and to make smoking more pleasant. The smoke from these products is a complex mixture of chemicals produced by the burning of tobacco and its additives. Tobacco smoke is made up of more than 7,000 chemicals, including over 70 known to cause cancer. Some of these substances are known to cause heart and lung diseases too, and all of them can be deadly. Environmental tobacco smoke. Is also called second hand smoke, because a portion comes from exhaled breath of the active smoker. This portion is called exhaled mainstream smoke which combined with sidestream smoke and smoke escaping from the cigarette rod. To make up environmental tobacco smoke. Tobacco smoke also contains tar and several toxic gases, carbon monoxide and nitrogen oxide. The specific chemical that produces the effect that people are looking for is nicotine, an additive drug. You might be surprised to know a larger proportion of many chemicals found in tobacco smoke are in the sidestream smoke. These chemicals are released directly into the environment. And also expose non-smokers to nicotine. We can measure this exposure in non-smoker by a specimens. The table lists some compounds that are present in higher amounts in sidestream smoke. For example, nicotine is higher in sidestream smoke. Than it is in the mainstream smoke that is inhaled by a factor of two or three. Cotinine is the major metabolite of nicotine and is a biomarker for exposure to tobacco smoke. Nicotine is quickly converted to cotinine in the body, but has a half life of up to 20 hours in the blood. Nicotine may also be introduced into the body through nicotine patches and chewing gum. These smoking cessation approaches will impact via monitoring data and need to be controlled for in tobacco smoke exposure studies through use of a study questionnaire to determine active use of these products. As we can see, cotinine has a methyl group, CH3. Then you'll see, later on, that that CH3 is converted to a adulterated compound with CD3 used as an internal standard in the essay. Cotinine can be measured in several bio specimens. Serum, saliva, urine and other samples are used to monitor for nicotine, particularly hair. Serum is collected invasively using a venapuncture to collect blood. Saliva is less invasive and can be self administered sample collection using a saliva sample kit. Saliva samples are stable at room temperature for several days. And can be easily mailed. Urine is also a non invasive sample, but is more difficult to mail. I've indicated on the bottom right diagram, the cotton plug which is used to chew on to collect saliva in the mouth, which is then placed into the sampling tube. Which is then sealed, and can be mailed. The distribution of cortinene levels in a populations provides an idea of what proportion are actively smoking, and those who are exposed only to environmental tobacco smoke. As we can see in this diagram, the two distributions split at approximately ten nanograms per mil of cortinene in serum. This is data from the CDC National. Health and nutrition examination survey. To measure cotinine levels in serum and saliva due to exposure from second hand smoke, it's necessary to have a sensitive analytical method based on mass spectrometry detection. With a detection limits of approximately 0.01 nano grams per mil of cotinine in serum or saliva. This graph shows that samples of saliva and serum obtained at the same time from a single donor are well correlated. And the studies using cotinine values measured in either of these biospecimens for exposure assessment may be compared. The slope is almost 1, and you can see there's very good correlation between the saliva cotinine sample and the serum cotinine sample. The analysis of cotinine in either saliva or serum uses the technique of Liquid Chromatography coupled to a triple quadrupole mass spectrometer. I will go through the steps necessary to process samples and measure the amount of cotinine in the bio specimen. Either saliva or serum. Are pre-treated and the cleaned extract is analyzed by this technique. As we can see 200 microliters of the serum or saliva is diluted with 2% ammonium hydroxide. And at this point we add 50 microliters of the deuterated internal standard that I mentioned earlier. This solution is passed through the well of a 96 well solid phase extraction plate which is shown at the top left of the slide. The cleaned extract is taken to dryness and ten microliters of the sample. That is being generated by dissolving an isopropynol is injected into the liquid chromatograph. The cotonine and the internal standards are separated from other components with the liquid chromatograph. And specific masses for cotonine and deuterated standard are selected, and the unique fragments detected in the mass spectrometer. Signals for cortinene are compared to the signals from the normal amount of internal standard added to the sample at the start of the sample preparation. Cortinene concentration is calculated from these responses. Detection limit is typically 0.01 nanograms per mil, using current [INAUDIBLE] instruments. This diagram represents the instrumental analysis of the same extracts, using this triple quadrupole mass spectrometer. The liquid sample extract is sprayed from a charged metal needle, imparting a positive charge to the mixture of molecules, which includes cotinine and the due directed internal standards. These positively charged ions can be selected based on their mass to charge ratio. And are accelerated through the three quadrupoles that make up the mass spectrometer. Each quadrupole acts like a tunable mass filter allowing only the selected mass to pass through. The characteristic mass for cotinine is 177 which corresponds to it's molecular weight plus 1. Which was hydrogen. The Q1 focuses these ions into Q2. Where specific fragmentation of the ion occurs as they collide with argon atoms introduced there. The characteristic fragments can be selected by their mass using Q3 and detected as a signal peak. The area of the peak. Is a function of the number and, or concentration of those molecules in the extract. Selecting the mass 180 for the tri deuterated internal standard allows its signal to be measured and compared to the cotinine signal. The calculation of cotinine resulting from second hand smoke exposure is then obtained. This diagram shows traces from each ion in the corresponding fragmentation as shown which indicates that both the coats name from the sample and the due to rated internal standards is present. The areas of the peaks are used to quantitative the level of cotinine in the sample and to confirm the presence. Of cotinine. You can see that the chromatigrams, all the peaks line up and the time is approximately the same for each stress. The cotinine levels were measured in two New York studies using this technique. The first one was the New York State Adult Tobacco Survey. This statewide study did not include New York City. When we evaluated the effect of the New York State Smoking Ban on restaurant workers, where we had a 104 participants and the nonsmoking general public, which we had almost 1600. The second study is the New York City Health and Nutrition Examination Survey called CHANES. Cotinine was a biomarker measured to assess smoking prevalence and public exposure to secondhand smoke in NYC. There were almost 1,800 participants, and levels compared with national data from the NHANES survey. The Clean Indoor Air Act is New York State's comprehensive law that went into effect on July the 24th 2003, prohibiting smoking in most public and private indoor work areas. Including bars, restaurants and bowling facilities. This law was intended to reduce exposure to second hand smoke among non smokers, and among employees who work in hospitality venues. New York State adult tobacco survey is an ongoing telephone survey to track smoking trends across the state. 2,000 random dialed calls each three months to assess the effectiveness of the New York State tobacco control program. Non smoking participants were asked to provide a saliva sample. If they agreed a sample kit was provided. That contained a consent form, sampling device and a mailer to return the specimen. The New York Adult Tobacco Survey therefore collected samples. These were independent samples from 2000. Residents each quarter. The survey generated state and regional information on tobacco use from adults aged 18 years and older. Nonsmoking participants from June 2003 through October 2004 were invited to provide a self-collected saliva sample. And you can see that out of almost 6,000. We ended up with about 6800 samples with efficient saliva to analyze for cotinine. In addition, we had 104 nonsmoking hospitality workers who provided saliva samples for this study. We used our LCME method with a detection of a 0.5 nanograms of ml saliva. This is the first report of a biologically validated population level reduction in second hand smoke among non-smokers after implantation of a comprehensive state. Smoking free air law. For the non-smoking hospitality workers the mean cotinine level decreased from a pre band level of 3.6 nanograms per mill. To 0.8 nanograms per mil of saliva cotinine 12 months after the ban went into effect, showing a significant drop resulting from the ban going into effect. The general population background, 0.35 nanograms per mil is a factor of ten less than the hospitality workers prior to the ban. So we can see that saliva cotinine levels in the general population of the nonsmoking. Adult tobacco survey participants decreased by 47% over the same period. These findings suggest that comprehensive smoking bans can reduce second hand smoke among non-smokers in the general population also. The slide gives more detail and shows the geometric mean levels of salivary cotinine among non smokers in the adult tobacco survey who submitted saliva samples. We already mentioned that they decreased by 47% from 0.78 milligrams per mil geometric mean during the pre ban period before the state law. Took effect and dropped 2.041 milligrams, mL, during the remaining period. The proportion of respondents with the Cotinine levels below the limit of detection increased from 32% to 52% when compared over the same period. That indicates that. Exposure was being reduced by the smoking ban over this period of time. So to summarize the health impacts of the New York state Clean Indoor Air Act, we see that non-smoking hospitalities worker exposure was reduced. The general non-smoker population exposure was also lowered. And this reduction. Coincided with the comprehensive ban going into effect. And so we see a direct response to the ban of smoking in public places. The second study was a New York City Health and Nutrition Examination Survey, CHANES. This was modeled after the CDC's national program, NHANES. Participants were interviewed and gave consent to have biospecimens taken for routine clinical tests. As well as for exposure assessment to environmental chemicals. To determine smoking prevalence and exposure to second hand smoke. A portion of the serum. Collective for other exposure measurements was tested for cotinine levels. These cotinine data were compared to the national averages. And if we look for the total number of participants we can see that for the New York city Haynes 23% of the participants were smokers, whereas in the CDC NHANES 29% were. And so we see there's less prevalence of smoking in New York City and that even goes as we split out males and female participants. So smoking prevalence is lower in New York City than nationally. Although New York City enacted comprehensive smoke free workplace legislation, CHANES data suggest that exposure to second hand smoke remains a significant health issue. Especially amount certain groups. And here we see that the Cotinine levels are somewhat higher in New York City 56.7% of elevated serum Cotinine in non smokers as opposed to 44.9% in the national survey. And this also holds for both males and females. The findings of higher prevalence of second hand smoke in New York City. Despite a lower smoking rate suggests that second hand exposure in dense urban settings may be a specific problem that we still need to deal with. So, just to summarize the New York Clean Indoor Air Act was signed on March 26th, 2003, prohibited smoking in virtually all workplaces in the state. It remains one of the strongest clean indoor air laws in the nation. Since 2003 exposure to second-hand smoke has largely been eliminated in all work places. The act has resulted in reduced exposure to second hand smoke among the general public and has direct. Measurable health impacts. The study in 2007 was estimated that, it prevented nearly 4,000 hospital admissions for heart attacks, in the first year following the law, saving New Yorkers direct health care costs of at least $56 million. Studies in other states and around the world have shown similar reduction in heart attacks and cost Validating the public health importance of reducing this type of chemical exposures. Thank you. [MUSIC]
