VITAMIN D DEFICIENCY IN FLIGHT ATTENDANTS AND PILOTS. Nicole Leonard

Transcription

1 VITAMIN D DEFICIENCY IN FLIGHT ATTENDANTS AND PILOTS by Nicole Leonard A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of University Studies In Nutritional Biology Approved: David Temme, Ph.D. Thesis Faculty Supervisor Ed Barbanell, Ph.D. Director, University Studies Program Martin Horvath, Ph.D. Honors Faculty Advisor Sylvia D. Torti, Ph.D. Dean, Honors College April 2015 Copyright 2015 All Rights Reserved

2 ABSTRACT Vitamin D is a curious molecule. While humans do get some vitamin D from their diet, most of it is synthesized in the body, so it does not fit the definition of a vitamin a molecule that must come from the diet. Vitamin D synthesis is initiated by the UVB ( nm) portion of ultraviolet radiation (UVR), and UVA ( nm) breaks it down. UVA and UVB have different properties, with UVA able to be transmitted through glass and plastics. UVB is blocked out by these materials, and is partially attenuated by the atmosphere depending on how much it has to pass through, varying most drastically by latitude and season (1). UVR also increases 15% with each 900 m (2,952 ft) increase in altitude (2). Recent studies have shown that the UVA exposure within the cockpit of a commercial airplane at a cruising altitude of 9,144 m (30,000 ft) is two times higher than at ground level (3). The average airline worker spends 75 hours a month flying, exposing them to conditions that theoretically increase their risk for vitamin D deficiency (4). It is known that airline workers have a higher prevalence of some health conditions (5), and that vitamin D regulates the expression of at least 900 genes in the human genome, many of which correspond to these conditions (6). My experiments tested how the environment of commercial flights affects the biochemistry of vitamin D precursors. I used UV spectrophotometry to analyze a solution of 7-dehydrocholesterol (7-DHC), a precursor to vitamin D found in the skin, before and after exposures to UVA, UVB, and flights. The light absorption properties changed post-exposure in all cases involving UVR. Different conditions, however, led to different changes. The differences in light absorption are due to a change in chemical structure in the solution, which provides further evidence that the in-flight environment could lead to chemical changes contributing to vitamin D deficiency in humans. ii

3 TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 16 RESULTS 20 DISCUSSION 27 ACKNOWLEDGEMENTS 31 DATA SUPPLEMENT 33 REFERENCES 37 iii

4 INTRODUCTION Vitamin D is a curious molecule. In humans and presumably in many, if not all, other animals, it is not even technically a vitamin based on the definition that vitamins are molecules organisms need that must be obtained from the diet because they cannot be synthesized. While there are some dietary sources of vitamin D including wild, fatty fish, fortified dairy products, and supplements, these sources are not usually sufficient for humans (with the exception of some traditional populations like the Inuit). Most of it must be synthesized through exposure to the sun. Vitamin D synthesis begins in the skin in the presence of a specific portion of ultraviolet radiation (UVR), with a derivative of cholesterol made in the body, and an inactive form is stored in the bloodstream and fat tissues. It is broken down by another portion of UVR. Because of the properties of UVR, its synthesis and degradation are affected by latitude, season, time of day, and local environmental conditions (7). Skin pigmentation likely evolved in response to vitamin D synthesis for this reason. While darker skin is more protective against DNA damage from UVR, it blocks vitamin D synthesis in places higher in latitude. People who migrated farther from the equator evolved to have lighter skin so that they could synthesize vitamin D for a longer portion of the year (8). The most well-known function of vitamin D is to regulate calcium absorption and deposition. Recent studies are finding that it is also involved with gene regulation of at least 900 specific genes including genes involved with cellular growth regulation and immune cell development (6). In general, signal molecules with actions similar to vitamin D tend to be regulated but always available. While there are inactive storage forms that are activated when the active form is needed, the variability of UVR does not guarantee 1

5 that sufficient supplies of the storage form are always plentiful. It is interesting that the concentration of a molecule with so many important roles is dependent upon how much sunlight the person is being exposed to. To complicate the issue even more, modern humans have moved to places that their skin is not necessary evolved to work optimally in and have increased their time spent in more unique UVR environments, including behind windows and inside airplanes that have even more of an impact on vitamin D production, contributing to the estimated one billion people that are deficient in the world (9). My thesis is divided into two parts. The purpose of the first part is to expand upon each of these aspects that make vitamin D so interesting to think about and how defects in its actions can lead to health conditions. The second part explains the experimental part of my project. I took samples of a precursor molecule to vitamin D into commercial airplanes to test the chemical stability in an environment created by modern humans that would theoretically have a negative impact on vitamin D levels of airline workers. Properties of Ultraviolet Radiation Ultraviolet radiation can be divided into different bands based on wavelength: UVA ( nm), UVB ( nm), and UVC ( nm). UVC is blocked out almost entirely by the earth s atmosphere, so it generally does not interact with life unless an artificial source is used (10). UVB is partially attenuated by the ozone, clouds, and pollution. The amount that reaches the earth is highly dependent on how much atmosphere it must pass through. The more oblique the angle between the sun and earth, proportionally less UVB penetrates to the earth s surface. This angle increases during the winter and with distance north or 2

6 south from the equator, so there is drastic variation seasonally, geographically, and even by the time of day. Higher latitudes get little to no exposure during the winter as the angle of those locations with the sun is too oblique and it is blocked by the greater amount of atmosphere that it must penetrate. There is also an increased exposure at higher elevations since there is less atmosphere between these points and the sun, and, at high enough elevations, the attenuation by clouds and aerosols is completely absent (10). UVR exposure increases as much as 15% with every 900 m increase in altitude (9). Artificial materials including glass, plastic, and clothing are able to block almost 100% of UVB, complicating the interactions between UVR and humans even more (1). UVA is able to pass through the atmosphere much more readily, so it defines most of the UVR that is experienced on earth. Intensity does not vary as much as that of UVB. It is still, however, the most intense at similar times of day and year as well as locations that UVB is at its highest. UVA is only partially attenuated by artificial materials. Depending on the material and thickness, up to 86% of UVA radiation is able to pass through windows. Researchers analyzing the environment within an office building found that 30% was able to pass through an average double-pane glass window, which means people sitting by those windows are constantly interacting with the radiation (1). Vitamin D Synthesis The synthesis of vitamin D (Figure 1) occurs in the skin starting with UVB radiation ( nm) causing the conversion of 7-dehydrocholesterol (7-DHC), or provitamin D, into previtamin D. Previtamin D is isomerized due to the heat at body temperature into cholecalciferol. This is absorbed into the capillaries of the skin and 3

7 transported to the liver via vitamin D binding protein where it is hydroxylated into calcidiol (25-hydroxycholecalciferol). Calcidiol is the inactive storage form that remains circulating or is stored in adipose tissue for later use. It is important that the active form is not constantly present because of the signaling pathways and gene regulation that vitamin D is responsible for. For it to be activated, one last hydroxyl group must be added, forming calcitriol (1,25-dihydroxycholecalciferol). In the classical case of calcium regulation, decreased blood calcium levels stimulate the parathyroid gland to release parathyroid hormone (PTH). PTH travels through the blood to receptors on the kidney that upregulate the enzyme 1α-hydroxylase, which will hydroxylate calcidiol. The newly formed calcitriol will enter back into circulation and cause increased absorption of calcium in the intestine and reabsorption in the kidney. 1α-hydroxylase can also be expressed locally in tissues where conditions stimulate the need for the active form to be present to modulate transcription in that specific cell or tissue. Because of its steroid structure, it is able to diffuse through cell membranes and bind to the vitamin D receptor (VDR) on the nuclear membrane (11). 4

8 Vitamin D Breakdown Vitamin D, in an inactive form, is stored in the blood and adipose tissue. Therefore, there is potentially a risk for vitamin D toxicity, or too much becoming active at the same time. Humans have evolved a few mechanisms help to reduce this risk. The first mechanism begins at the level of synthesis and is controlled by UVR. Once previtamin D isomerizes into cholecalciferol, it must be absorbed into the bloodstream. If local concentrations in the capillaries of the skin are too high in cholecalciferol, meaning a lot has been recently produced, vitamin D binding proteins will be saturated and unable to transport more until some of it has been processed and used. Cholecalciferol is broken down into 5,6-trans-vitamin D, suprasterol 1, and suprasterol 2, all inactive byproducts, if it is exposed to too much UVR before being absorbed in the bloodstream. These molecules are simply metabolized and recycled. Alternatively, if the concentration of cholecalciferol begins to build up in the skin, the previtamin D will initially isomerize into tachysterol or lumisterol instead. This delay in absorption in the blood allowing for a buildup of cholecalciferol and the isomerization of previtamin D into different chemicals could have been positively selected for as a protective measure against vitamin D toxicity during times of high UVR exposure. Another UVR driven mechanism involves a different portion of UVR from synthesis: UVA ( nm). Since UVA is able to penetrate farther into the dermis with its longer wavelengths, it breaks down vitamin D and precursors that are either in the skin or circulating through capillaries just under the surface of the skin (1). The difference between breakdown caused by excess UVR versus by UVA specifically is that when there is excess radiation in general, there is already a sufficient amount of vitamin D produced. UVA depletes the stores that are already 5

9 circulating and prevents more from being made by also breaking down the precursors that have not yet been absorbed or converted to the next step. All signaling molecules must have a way to be turned off. Some are isolated into vesicles while others are broken down. The presence of calcitriol also turns on the local expression of an enzyme called 25-hydroxyvitaminD-24-hydroxylase (24-OHase), which converts calcitriol into calcitroic acid so that it will stop signaling until that signal is needed again (11). Unlike being controlled by UVR like the previously mentioned mechanisms, this depends on the presence of calcitriol itself. General Mechanism of Gene Regulation by Vitamin D The vitamin D receptor (VDR) is located on the nuclear membrane and is activated by the binding of vitamin D. The receptor forms a heterodimer with retinoid X receptor and together they bind to vitamin D response elements (VDREs) along the DNA. These interactions can control whether the corresponding gene is expressed or not. There are at least 2,776 of these sites throughout the human genome. Examples of genes affected include IRF8 and PTPN2 which are associated with cell differentiation and immune system function. When these genes are not properly regulated, they can lead to the development of multiple sclerosis or Crohn s disease (9). Melanin and Skin Pigmentation Patterns Humans were thought to have first evolved in the equatorial region of Africa. This means that the ancestral state would have been exposed to high intensity UVR yearround. Other primates in the same area had dark, pigmented fur to protect themselves from UVR-induced DNA or skin damage. Humans, on the other hand, lost most of this protective fur. In order to live there successfully, they had to compensate for this loss of 6

10 protection in some way. Presumably, this was accomplished by the deposition of the pigment found in the fur of other animals in their skin. This pigment, called melanin, protects the skin both by absorbing much of the UVR and neutralizing reactive oxygen species generated by oxidative damage from the remaining UVR. In essence, it is a natural sunscreen. There are two forms of melanin produced in humans: a reddish-yellow pheomelanin and a black-brown eumelanin. Eumelanin is responsible for making darker skin, increasing the absorption of UVR, thereby making it more protective. Therefore, the first human ancestors likely had very dark skin pigmented by eumelanin to protect them from the constant high exposure to UVR (12). Skin does not only have to be protective against the sun, but also against physical interactions between the body and the world around it. It is made up of multiple layers of cells, varying in characteristics. The outside layer is made up of dead cells, so that when they are rubbed off or harmed, there is little to no consequence. The cells of the skin are reinforced by a structural protein called keratin to make them more durable. Keratin is produced in keratinocytes, which are the most common cell in the epidermis (13). Given their abundance, it would make sense for melanin to be produced in these cells. Keratinocytes, however, lack the ability to produce melanin, perhaps because they must put their resources towards keratin production. Instead, specialized dendritic cells scattered throughout the lower epidermis called melanocytes are responsible for melanin synthesis. Melanocytes contain unique organelles called melanosomes, and it is within these vesicles that melanin synthesis occurs. Melanosomes must go through four stages of maturation. They start their progression through these stages between the nucleus and Golgi apparatus of a 7

11 melanocyte. They receive enzymes required for melanin synthesis and eventually become saturated with the pigment (12). Once completely saturated, they are exocytosed by the melanocytes and taken up through endocytosis by keratinocytes. The keratinocytemelanocyte pair is called the epidermal melanin unit. Once the melanosomes are transferred to the keratinocytes, the vesicles full of melanin initially collect above the nucleus to form a physical barrier between the DNA and UVR, reducing the chance of mutations caused by oxidative damage from the UVR. Once there is sufficient protection over the nucleus, they begin to spread throughout the rest of the cell, resulting in darker skin (10). The UVR environment became more complicated as humans migrated away from the equator. Not only does UVR intensity tend to decrease as distance from the equator increases, but also the overall pattern of UVR intensity with latitude varies across seasons. Given this variation, maintaining the same dark pigmentation presumably found in equatorial humans could cause problems in vitamin D synthesis so much of the available UVB would be absorbed by the pigment that there wouldn t be enough to initiate the synthesis pathway (8). This leads to an interesting idea: much of the variation in skin color in humans is a vitamin D story. By reducing the amount of pigment produced, humans at higher latitudes would be able to synthesize more vitamin D. This suggests that lighter colored skin was selected for in these locations. This change was not caused by a change in the number of melanocytes (humans all have about the same amount), but rather by mutations in the three to four genes that control interactions within the epidermal melanin unit. The details are not exactly known, but the genes likely control how much melanin is able to be 8

12 deposited in the keratinocytes (10). The loss of some protection due to lightening of the skin isn t dangerous as long as the person lives in an area with weak enough UVR. So, the extent to which melanin production decreased was dependent on each given location. This explains how, for example, people with very light skin can live in cloudy places at higher latitudes without excessive skin damage (10). Because of the seasonality of UVR, humans also evolved a tanning response, or variation in skin pigmentation relative to the intensity of UVR at a given time. If they maintained the same color skin throughout the whole year, they would either not be protected enough during the summer or not able to make adequate vitamin D during the winter. Melanin production is affected by chemical signals resulting from cellular stress mainly at the level of the melanocyte. When melanocytes detect UVR, proopiomelanocortin (POMC) is upregulated, which will be converted to melanocytestimulating hormone (MSH), and the receptor for MSH, MC1-R. MC1-R is a G proteincoupled receptor that leads to the initiation of melanogenesis. Tyrosinase enzymes involved in melanogenesis are also expressed at higher levels, and other cellular signaling cascades involving protein kinases begin to activate. These events lead to the progression of melanosomes through the four stages to maturation. Keratinocytes are also able to detect UVR and produce POMC and endothelin-1, which are paracrine signals that act locally on nearby melanocytes. These chemicals cause various changes in the structure and function of melanocytes and other cells in the skin, and eventually lead to an increased transfer of melanosomes to keratinocytes (10). These pathways highlight the importance of the epidermal melanin unit being able to work together and respond to 9

13 similar stress factors in a complementary way to cause fast and effective changes in order to best protect the cell. Given that the angle between the sun and places far north and south is too oblique during the winter months to generate any UVB exposure, the people there do not have enough UVB to synthesize vitamin D no matter how light their skin is (12). This is a complex issue that is not easily explained. There are a few potential solutions to the problem. First, they may have more dietary sources that provide them with adequate supplies to get them through the season. Fatty fish and mammals tend to live in these regions, so perhaps they can get enough by eating the animals vitamin D stores. Their genes may also be adapted to respond to the lack or presence of vitamin D to switch between winter and summer modes, so they may be able to survive in a way that they don t need to rely on it for some of its functions during that time because the genes may be regulated in different ways. The regulation of melanin production is an effective system in balancing protection against UVR with the ability to synthesize vitamin D, but the skin needs time to keep up with gradually changing conditions. As UVR steadily increases from the winter to the summer, melanin production, theoretically, also increases at the same rate. By the time UVR is most intense, larger amounts of melanin are also present so that the person is protected but still able to make vitamin D. With the modern means of travel, people go to places that their skin is not adapted to and this intermittent exposure to UVR of a different intensity than the skin is prepared for can put them at risk for things like skin cancers or vitamin deficiencies, depending on which way they are going. Even if people are always in the same geographical location, the windows that they sit behind for 10

14 a majority of their life creates an unnatural UVR environment for the skin, and melanin production is not able to keep up with seasonal changes properly (12). Populations at a Higher Risk for Vitamin D Deficiency Based on the properties of UVR and the relation to vitamin D synthesis and breakdown, those who spend the most time behind windows or other materials that only transmit UVA are the most likely to develop a vitamin D deficiency. Godar and colleagues discuss how the UV barrier created by humans with increased use of buildings and cars essentially excludes UVB from our lives because many people work from dawn until dusk, and by the time they get outside there is very little, if any, UVB reaching these people. To make matters worse, a greater exposure to UVA destroys any vitamin D circulating in the blood that may have been previously synthesized on the weekend or during a vacation. They hypothesized that outdoor workers maintain a relatively stable and adequate vitamin D concentration when compared to the drastically changing, mostly inadequate levels for indoor workers (1). The Unique Airplane Environment People don t usually think they are entering a very unique environment when they take off in an airplane; an environment that could have different biological effects on them than anywhere else. Airplanes are essentially office buildings, only much closer to the sun. Given that UVR increases with altitude, it would makes sense that there is an even higher risk of vitamin D breakdown in-flight than sitting at an office desk at sea level. A recent study measured UVA exposure in the cockpit of an airplane at cruising altitude (30,000 ft) and found it to be twice as high as at ground level. While flight attendants aren t subject so such direct exposure as pilots are sitting in the cockpit, the 11

15 UVR coming through the cabin windows is still filtered to exclude UVB and only transmit UVA, and it is more intense than a similar situation at ground level (3). The average airline worker spends about 75 hours a month flying (4), so this unnatural exposure could easily add up over time to contribute to a deficiency in vitamin D. Data collected on the health status of airline workers supports the idea that the environment they spend a large amount of time in has biological effects and could contribute to increased prevalence of many diseases when compared to the general population. The following diseases are more prevalent in flight attendants in relation to job tenure than the general population: chronic bronchitis, cardiovascular disease, skin cancer, hearing loss, and depression/anxiety (5). These findings are especially interesting because many risk factors for some of these diseases such as smoking and hypertension are decreased in the flight attendant population, suggesting that something else plays a role in the development of these conditions (5). Relationships between Vitamin D and Flight Attendant Health Conditions Relationships have been found between vitamin D and a vast array of diseases. Interestingly, each of the conditions found at high rates in flight attendants relative to the amount of time they have been working has associations with an increased risk due to vitamin D deficiency. Bronchitis is an infection of the lower respiratory tract, usually associated with smokers in the development of chronic obstructive pulmonary disease or complications due to asthma or allergies, yet flight attendants have a significantly lower prevalence of all of those risk factors (5). VDRs are located in the cells lining the respiratory tract, and can cause the expression of antimicrobial molecules. When secreted locally from these 12

16 cells, these molecules can help protect the body against infection (15). Since other risk factors are not present, the higher prevalence is likely contributed to by a vitamin D deficiency. Cardiovascular disease (CVD) is the number one killer in the United States. This category includes peripheral vascular disease, diabetes mellitus, metabolic syndrome, coronary artery disease, and heart failure (16). Hypertension is generally a risk factor for the development of these conditions, but again, flight attendants tend not to experience this (5). Calcification of arteries, or atherosclerosis, is also a major contributor to CVD. Negative correlations between vitamin D levels and atherosclerosis have been found in multiple studies, although the direct mechanism is unknown. One known mechanism contributing to arterial dysfunction relates to inflammation of the arterial walls. Inflammation increases the chance that cholesterol, platelets, calcium, and other particles in the blood stream will aggregate and cause a blockage. Vitamin D has immune cell targets regions that regulate inflammatory cytokine production by encouraging the differentiation into antiatherogenic TH2 cells and down regulating the production of inflammatory signals (16). If there is not enough vitamin D present, inflammation would likely be higher and could lead to increased plaque development. Skin cancer, melanoma in particular, is among the top five most prevalent cancers in the US and is on the rise (17). Melanoma is a cancer of melanocytes, the cells that were previously described to produce melanin. Melanoma cells have a VDR on their nucleus, and the binding of vitamin D triggers growth inhibition or apoptosis through modulating transcription of necessary proteins (1). Interestingly, the same receptor being activated prevents apoptosis in normal melanocytes (1). The same mechanism can control 13

17 various other types of cancers as well. What is more unique to melanocytes and keratinocytes, however, is that both have the enzymes necessary to add hydroxyl groups to cholecalciferol and calcidiol like what happens in the liver and kidney (1). This suggests a tight connection evolutionarily between vitamin D and those cell types because these genes are not transcribed in all cell types. Hearing loss could easily be attributed to the loudness of the engines constantly running while airplanes fly, but an alternative explanation connects to vitamin D. Hearing involves small bones of the ear called the malleus, incus, and stapes, moving in response to sound waves. Just as vitamin D deficiency is known to cause bone weakness in general because of a lack of calcium absorption and deposition, these bones are also at risk of weakening due to a deficiency, especially with overuse. Calcium-dependent functions of the cochlea, the sensory portion of the inner ear, may also be disturbed if not enough calcium is present, further exacerbating hearing impairment (18). The exact connections between depression and vitamin D are still not completely clear because of the complexity of depression. Some would argue that depression causes people to stay inside, which would limit their ability to synthesize vitamin D and make depression the cause of the deficiency rather than vice versa. Many studies have shown, however, that vitamin D supplements can help reduce or cure depression, and that deficiency correlates to depression (19). VDRs have been found in the human brain, including in regions that are modified in depressed people. Vitamin D is thought to be able to modulate neurotransmitter production and have paracrine effects. Just like in cells of the skin, cells of the brain also contain converting enzymes and are able to continue 14

18 the synthesis of vitamin D from inactive precursors, again suggesting that there is indeed an importance of the molecule in the brain (19). Summary Vitamin D has a complex relationship with UVR, and the modern day lifestyle makes it even more complicated (1). People evolved to have certain skin colors based on where they lived, but now people regularly move to different places, increasing their risk of either damage from too much UVR or a vitamin D deficiency from too little (12). Much more time is spent behind windows and inside airplanes, which can further modify the risks. As shown in studies on flight attendants and pilots, health conditions that relate to malfunctions in the typical roles of vitamin D are increased in populations that spend too much time in these environments (5). The roles of vitamin D and factors that affect the supply of it within the human body are currently big topics in research as more roles are discovered. The purpose of the experimental part of my thesis was to contribute to the knowledge about how different conditions may affect vitamin D precursors, particularly in the main cabin of commercial airline flights. Previous studies on flight attendant and pilot health have not proposed the idea that vitamin D could be a major factor contributing to the problems they face. Based on Godar s study on office workers (1) and physical properties of UVR, I hypothesized that the in-flight environment would alter the chemical structure and properties of 7-DHC, the first molecule in the vitamin D synthesis pathway. 15

19 METHODS Experimental Design UV spectrophotometry was used to analyze the composition of a solution of 7- DHC. Quartz cuvettes containing 3 ml of the solution were first exposed to a UVB lamp simulating the first steps of vitamin D synthesis, which were then further exposed to various conditions including windows, natural sunlight, a UVA lamp, and commercial airline flights. Absorption spectra were taken before and after exposures. The starting solution was made up of 50 ug/ml 7-DHC in isopropyl alcohol (91%) as Sternberg and colleagues used in their study (20). Other studies using the same method used methanol or ethanol as the solvent (6, 19), but due to regulations set by the Transportation Security Administration (22), I chose to use isopropanol. The 7-DHC was purchased through Sigma-Aldrich and was stored at their recommended -4 degrees Celsius. The isopropanol was purchased at a local grocery store and stored in the lab at room temperature. Exposures I first obtained a baseline spectrum of the 7-DHC solution before exposure. I then exposed different samples from the stock solution to different environments (Table 1). Table 1: 7-DHC exposure conditions Condition UVB Lamp UVA Lamp- 265 nm Lab Bench (no windows) Outside, 4,200 ft elevation, sunny Behind window, chemistry building Behind window, house Exposure Time 45 min, analyzed every 5 min 2.5 hr 0.5 hr, analyzed every 5 min 3 hr 0.5 hr, analyzed every 5 min 3 hr 16

20 To simulate some vitamin D synthesis prior to exposure to other conditions, I exposed samples that had each been previously exposed to the UVB lamp for 45 minutes to different conditions. Table 2: 7-DHC exposures after UVB exposure Condition UVA Lamp- 265 nm Outside, 6,800 ft elevation, sunny Behind window, 4,200 ft elevation Behind window, 6,800 ft elevation Airplane, SLC-ATL Airplane, SLC-DTW Airplane, SLC-PHX Airplane, PXH-SLC Airplane, JFK-SLC Airplane, ATL-SLC Airplane, DTW-BNA Airplane, BNA-ATL Airplane, ATL-BHM Exposure Time 1 hr, 2 hr, 3 hr 3 hr 1.5 hr, 3 hr 3 hr 4 hr 3 hr 1.5 hr 1.5 hr 5.5 hr 4.5 hr 1.5 hr 0.5 hr 0.5 hr Exposure times were based on flight time or logistics. For those with multiple times listed, each time represents a different sample. The exceptions are the conditions that state analyzed every 5 min, meaning spectra of the same solution were taken over 5 minute increments for the duration of the exposure. These tests were done in close proximity to the spectrophotometer, therefore such measurements were possible. 17

21 Data Analysis A Shimadzu UV-1800 UV spectrophotometer was used and each sample was scanned between 230 and 330 nm at 1 nm steps using UVProbe2.33. Data was collected in UVProbe as.spc files and downloaded as a text file to be imported into Microsoft Excel when the graphs were made. Scattering Effect To control for the potential scattering effect potentially due to other molecules or particles in solution that was seen in the analyzed samples, a Savitzky Golay filter was applied to the data in Microsoft Excel. It was designed to take the second derivative of the graph in order to minimize the effect on the data for comparisons (23). The second derivative graphs are located in the data supplement (Figures 7b-11b). Transportation and Storage of Samples The 50 ug/ml stock solutions were stored in 60 ml amber glass bottles and each individual sample was stored in a 15 ml amber glass bottle before and after it was put into the cuvette for exposure. The samples in the lab were kept in a 4 Celsius refrigerator, and the samples that were taken out of the lab were kept on ice in an insulated container. Stability of Unexposed Solutions over Time I tested samples at different time intervals after exposure to one of the test conditions to determine the stability over time being stored in the refrigerator, as well as the stock solution that I drew my samples from (Figure 12-data supplement). 18

22 Stability of Solvent I exposed separate samples of isopropyl alcohol without any added chemical to UVA and UVB light for 2 hours to determine the stability of the solvent itself. I also collected data of the isopropyl alcohol straight from the bottle at the same location and temperature that it had been stored at for the duration of my project: F23 Celsius under fluorescent light in a translucent white plastic bottle (Figure 11a, 11b-data supplement). Centrifugation The high absorbances in many of the samples suggested that large molecules, particles, or bacteria could have been in the samples. In an attempt to see if there was contamination, I centrifuged the samples and reanalyzed the solution from the upper portion of the Eppendorf tubes. There were no changes, suggesting that they likely contain large molecules that could have been synthesized through an internal polymerization reaction. 19

23 RESULTS Ground Level UVR Exposures According to the literature, there should have been different changes in the vitamin D precursors when exposed to UVA versus UVB. The data in Figures 4 and 5 show that while UVB appears to have a progressive effect on the 7-DHC, showing gradual yet uniform changes in the solution with time, UVA caused seemingly random and drastic changes. There were no changes over 30 minutes of the sample sitting under fluorescent lighting on the lab bench (Figure 5), showing that it must indeed be UVR and not just light in general that causes chemical changes. The sample that was placed behind a window (Figure 6) underwent small changes. The exact window material and thickness are unknown, but it was done in the late afternoon during the winter when UVR is at its lowest, so large changes should not have been expected as with the direct, controlled UVR sources. Baseline Figure 2: The original spectra of 7-DHC before any exposure. 20

24 Incremental Changes The following graphs (Figures 3-7a) were made to show data taken incrementally under different conditions. UVB Absorbance Wavelength (nm) 0 min 5 min 10 min 15 min 20 min 25 min 30 min 45 min Figure 3: 7-DHC every 5 minutes during exposure to UVB lamp. This simulates the first step of vitamin D synthesis that occurs in the skin. Figure 4: 7-DHC was previously exposed to UVB, and this graph shows how it changed after each hour of exposure to the UVA lamp. 21

25 Figure 5: 7-DHC every 5 minutes while sitting on the lab bench under the fluorescent room lighting Window Absorbance Wavelength (nm) 0 min 5 min 10 min 15 min 20 min 25 min Figure 6: 7-DHC every 5 minutes during exposure to a window in a building at the University of Utah on a winter afternoon. 22

26 SLC DTW Flight Absorbance Wavelength (nm) 0 min 45 min 90 min 180 min Figure 7a: The 7-DHC was previously exposed to UVB, and this graph shows how it changed over the duration of a flight between Salt Lake City, UT and Detroit, MI during the winter. Flight Samples and Comparisons Figures 8a-10a show representative samples of results from the flights and how they compare to theoretically similar conditions. Included are both the original spectra and the second derivatives in order to compare more accurately. The original spectra display a scattering effect, in that the slopes of the curves overall are due to light scattering in the samples and may not be a true relative representation of the data. See Figures 8b-10b in the data supplement for the second derivative graphs. Post-flight analyses resulted in a variety of different shaped spectra (Figures 7a- 8b). After some flights, the spectrum resembled a similar shape to the original 7-DHC spectrum but with different major peak heights. Others seemed to experience random, drastic changes as seen in the spectra resulting from UVA exposure. Based off of the progressive nature of the spectra in Figure 7a, it seems as if the longer the sample is 23

27 exposed, e.g. the longer the flight, the greater the change would be. This trend is maintained by most of the flights shown in Figure 8a except the shortest flight, ATL- BHM, which showed the most drastic change. There were no other trends between factors including direction of travel, time of day, or type of aircraft that related strongly to the wide variation between results. I do not know how to explain this. Figures 9a-9b show one sample that changed in a similar way to a sample that was placed behind a glass window for the same amount of time as the flight lasted (1.5 hours). Figures 10a-10b show another sample that resembled a change after UVA for the same amount of time (3 hours). Again, the variation between the samples from different flights is hard to explain, but there were samples that changed in similar ways to ground level controls that have been previously been proven to induce changes in vitamin D synthesis (1). Figure 8a: These are examples of resulting spectra post-flight. Various patterns were seen. The legend is written with the airport codes, origin followed by destination. All samples were exposed for the flight duration, so exposure times vary. 24

28 Figure 9a: This graph compares the changes seen between the sample sitting behind a window at approximately 4,200 ft and behind the window of an airplane at approximately 30,000 ft. Both samples were exposed for 90 minutes. UVA vs. Airplane Absorbance Wavelength (nm) Pre exposure Airplane UVA Figure 10a: This graph compares the changes seen between a three hour flight and three hour exposure to the UVA lamp in the lab. 25

29 Solvent I was told that isopropanol could be light reactive, so I tested the solvent without any added chemical under some of the same conditions that I tested the chemical under. Due to time and logistical constraints, I was only able to expose it to UVA and UVB lamps. Isopropanol underwent a change in chemical light absorbing properties after exposure to both UVA and UVB sources (Figure 11a, 11b in data supplement). Isopropanol Absorbance Wavelength (nm) Pre exposure UVA UVB Figure 12a: This shows the spectra of isopropanol during storage conditions and after exposure to UVA and to UVB. 26

30 DISCUSSION Summary of Results Each condition tested resulted in a change in light absorbing properties. The properties changed because of a change in chemical structure within the solution. Changes post-flight varied in the shape of the spectra, but there were examples that resembled patterns seen after controlled ground level UVA exposure and after exposure to ground level windows. Comparison to Other Studies The original spectrum from 7-DHC before any exposure (Figure 2) matched what was published in Terenetskaya s study (21) that used the same method with ethanol instead of isopropanol. The changes after exposure to UVB light (Figure 3) had the same patterns in terms of changes in the shape of the curve at specific peaks, but did not happen to the same extent, perhaps due to the strength of the UVB source. Solvent Almost at the end of my data collection, it was brought to my attention that isopropanol is generally stored in lightproof containers due to potential UVR sensitivity. The tests done with the UVA and UVB lamps on isopropanol without 7-DHC showed that the spectrum of the solvent did indeed change, suggesting that it is affected by UVR and could have interfered with the other data collected. This did, therefore, have had an effect on the spectra post UVR exposure even in the samples that contained 7-DHC. In retrospect I should have done the solvent test before progressing with the rest of the tests. The spectrum of the isopropanol from its storage container did not show any absorbance, which suggests that even if the solvent changing in my experiments 27

31 interferes with my data, the environment of the airplane did contain some kind of exposure stronger than what is produced by fluorescent lighting that is able to affect chemical properties (Figure 11a, 11b). Accounting for Time It could be argued that it was the time between original and post-exposure analyses that caused or allowed for a change in the solution to occur, but the stability of a variety of the solutions after being retested at different time intervals helped to rule this out. They remain relatively the same upon retesting on a later date, so long as the samples were protected from UVR. The small changes visible are not progressive, as in the first sample is not highest and the last sample lowest at a given peak, rather it appears to be more random. Since the changes between the pre- and post-exposure analyses were far more extreme than the small changes after the passage of time without new exposure, it was mostly the UVR exposure that induced the change (Figures in data supplement). Limitations There are several limitations to this study. Given the very complex environment within a flying airplane, it is very hard to isolate what it is that caused the chemical changes. While there has been shown to be UVA radiation in airplanes, they are also known to have cosmic/ionizing radiation and are in constant communication through the use of radio waves. Countless other contributing factors could include the cabin pressurization, electronic controls in the cockpit, the use of the engines, etc. It is impossible to control for each variable unless an artificial yet identical environment is created where each thing can be regulated separate from the others, but the recreation in a 28

32 controlled UVA environment at ground level suggests that it is indeed the UVA that contributes to the chemical changes. A problem with the data I collected is that, with the resources I currently have, I cannot determine what is actually in the samples after the exposures. Many of the spectra were very unique and it is impossible for me to explain exactly what happened in each case. Other methods such as nuclear magnetic resonance spectroscopy or gas chromatography may give more of a sense of at least the properties of what is present in solution, but it is hard to assign changes in peaks to specific molecules when dealing with a reaction that has many different possible pathways as seen in the discussion regarding vitamin D synthesis and breakdown. Further Study Ideas Another way to realistically study vitamin D levels in airline workers would be to test them before and after flights using a test that can detect concentrations of calcidiol in the blood. The change, if any, could be compared with the same type of test over the same time duration but under different environments such as outside, inside a building with windows, and inside a building with no windows. Unfortunately I did not have the time or resources to do such a study, but it could provide much more reliable data than using a solution to simulate what could be going on in the body. For a slightly different perspective on vitamin D, a latitudinal study using animals native to certain areas could be conducted. Blood samples could be tested for vitamin D levels and compared to similar animals at different latitudes. For example, hooved mammals from various regions could be studied and the samples could be taken during different seasons to compare the vitamin D status both to similar animals in different 29

33 places and within the same animal seasonally. Humans would be difficult to use for this because they often don t live where their ancestors evolved, so results may not tell a traditional story of vitamin D status in humans. It could, however, still provide an interesting contribution to the ongoing research on this important topic. 30

34 ACKNOWLEDGEMENTS The reason that I got interested in this topic and was so motivated to study it was that my mom is a flight attendant and was diagnosed with melanoma a few years ago. Luckily it was removed before metastasis, though. When I first found out she had it, I really didn t think enough of it because I was unaware of the high risk of death. I happened to be taking Biology 3380, Evolutionary and Physiological Basis of Health, the semester after it happened, where I began learning about the story of melanoma. We talked about how rates are high in indoor workers, and it got me thinking that the same explanation could translate to airline workers. I asked my mom if she knew if rates were high in her profession and she said she thought they were. A search of the literature on flight attendant and pilot health, which I was surprised to find so much of, confirmed my prediction and her guess. I could not find any studies that suggested that vitamin D contributes to the issue in this population, which is the gap that I hoped to fill in. While my data may not have been strong enough to support my hypothesis, I still believe there is a chance for meaningful correlation and further studies should be done with more time and resources. Thank you, Mom, for being my inspiration to take on this project and providing me with an intrinsic motivation to investigate in an attempt to prevent this from happening to your friends and coworkers. I would also like to thank the following people: Dr. Dave Temme for being the faculty sponsor and major inspiration for my project. I got the idea for this thesis in his class and he supported me in helping to get funding to move forward with the project. Without having taken Biology 3380 or his other classes, I would probably not have been able to link these ideas together in the way 31

35 I did. He really changed the way I think about biology and any other subject I encounter, and that has most definitely changed my learning experience throughout my undergraduate career. Dr. Jennifer Heemstra and her lab group for letting me use the spectrophotometer in her lab at the University of Utah. At the start of my project, I had no physical resources. I asked around the chemistry department to find someone with the machine I needed to look at my data, and she welcomed me into her lab. It was very much appreciated and I would not have been able to do any analysis without this opportunity. Dr. Martin Horvath for assisting me with data analysis and giving me pointers to be able to finish up my project. He was very positive and motivated me to continue on, even when my data was becoming hard for me to analyze; I was really lucky to connect with him when I did. Funding and Resources Funding for this project was provided by the Undergraduate Research Opportunities Program at the University of Utah. Without the money they were able to provide for supplies including my chemicals, cuvettes, and amber bottles, I would have been far more limited in what I could have done. Aside from just monetary support, they also sponsored lectures on many aspects of the research process, which made it much easier to make progress. 32

36 DATA SUPPLEMENT Second Derivative of SLC DTW Flight Absorbance min 45 min 90 min 120 min Wavelength (nm) Figure 7b: The second derivative of the graph in Figure 7a. Figure 8b: The second derivative of the graph in Figure 8a. 33

37 Second Derivative of Window vs. Airplane Absorbance Pre exposure Window Airplane Wavelength (nm) Figure 9b: The second derivative of the graph in Figure 9a Second Derivative of UVA vs. Airplane Absorbance Wavelength (nm) Pre exposure Airplane UVA Figure 10b: The second derivative of the graph in Figure 10a. 34

38 Second Derivative of Isopropanol Absorbance Wavelength Pre exposure UVA UVB Figure 11b: The second derivative of the graph in Figure 11a DHC Stock Solution Absorbance Wavelength (nm) 11/20/ /8/ /19/ /2/2015 Figure 12: This shows samples taken directly from the stock solution on four different days (indicated in the legend) during the span of data testing. 35

39 Changes in Post Flight Sample over 3 Days Absorbance Wavelength (nm) Post Flight Post 3 Days Figure 13: This graph shows short term changes in samples post-flight. It represents the time between initial exposure and testing of the sample. The data above an absorbance of 3 is not exactly the same, but at an absorbance that large the details are not as reliable. The overall shape and pattern stayed constant after 3 days of being stored at 4 degrees Celsius Changes in Post Flight Sample over 2 Weeks Absorbance Wavelength Post Flight Post 2 Weeks Figure 14: This graph shows longer term changes in samples post-flight. A different post-flight sample was used than in Figure 14 to show universality amongst the samples in stability over time. There were small changes, but mostly at higher absorbances. The overall shape and pattern remained constant after 2 weeks of being stored at 4 degrees Celsius. 36