Introduction Lung Cancer (lc) Is One Of The Most Common

INTRODUCTION Lung cancer (LC) is one of the most common form of cancers world-wide (1) and continues to be the single greatest contributor of cancer-related mortality in the USA in both males and females (1,2). Currently, in the United States alone, more than one third (40%) of the adult population has an increased risk of developing LC due to current or former cigarette use (2).

Cigarette smoking is the main risk factor for developing

primary lung cancer (PLC). Former smokers, even years later remain at a higher risk for PLC compared to individuals who have never smoked. Smoking cessation, however, is the most effective means of reducing the risk of LC. However, many questions remain regarding why only some heavy smokers develop LC. Nutritional status and dietary habits may account for a significant proportion of this variability in risk. Therefore, investigations of the nutritional status and the expression of nutrient-related intermediate end point biomarkers of lung cancer may provide valuable information on reducing the risk of developing LC or its early detection. Several studies indicate that exogenous and endogenous factors interact and modify the risk of developing LC in smokers (4,5, 6,7,). Smokers are more likely to have less healthful diets, which include lower intake of fruits and vegetables ( 3). Dietary intake of fruits and vegetables provide antioxidant micronutrients such as vitamin C and precursors for vitamin A (beta-carotene) and one carbon nutrients such as folate. These micronutrients and other antioxidant micronutrients (example, vitamin E) and other one carbon nutrients (example, vitaminB12) have been hypothesized to reduce LC risk because of their roles as regulators of cell differentiation (vitamin A) ( 4), free-radical

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scavenging (vitamins C ,E and beta carotene) (5) and modulators of DNA synthesis, DNA repair or DNA methylation (folate and vitamin B12 (6, 7). Because not all smokers are able to quit smoking completely or even reduce the number of cigarettes they smoke, it is important that patients be informed of other factors which may modify their risk for development of PLC. In addition to avoiding cigarette smoking, the adoption of a healthier diet which provides “cancer-protective” micronutrients is likely to be one of the most relevant factors for reducing the risk of smoking-related PLC (8). A number of case-control studies and prospective cohort studies have shown that higher consumption of fruits and vegetables is associated with a reduced risk of LC. Most of these studies have focused on identifying the specific components in fruits and vegetables and its effect on LC risk. Several cross-sectional observational studies have focused on antioxidant nutrients specially β -carotene and reduced risk of PLC. Circulating concentrations of total carotenoids or specific carotenoids have been associated with reduced risk of LC. Several of these observational and intervention studies have examined the isolated effects of several micronutrients (folate, ß-carotene, vitamins A, B-12, E, C or ß-carotene) in relation to PLC. To our knowledge, however, there are no published reports that evaluated a panel of circulating concentrations of micronutrients in relation to risk of smoking related PLC. Therefore, evaluation of associations among circulating concentrations of micronutrients and lung cancer risk will make a significant contribution to the understanding of their roles in the prevention of smoking related cancers of the lung. The purpose of this study was to determine the association between a panel of selected circulating concentrations of micronutrients (plasma and red blood cell - folate, vitamins A, B-12, E, C, and β-Carotene) and the risk of being diagnosed with smoking

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related PLC, after adjusting for established risk factors for LC reported in the literature (e.g. age, race, gender, and alcohol consumption). Research Hypothesis and Specific Aims The overall research objective of the study was to evaluate associations between micronutrient status and the likelihood of being diagnosed with smoking related PLC. Hypothesis Circulating concentrations of micronutrients (folate, vitamins B-12, A, E, C and β carotene) will be associated with the risk of being diagnosed with PLC among smoker after adjusting for age, race, gender and alcohol consumption. Specific Aims 1. Measure circulating concentrations of folate (both plasma and red blood cell) by the L.Casei microbiological assay, vitamin B-12 by a competitive radio- binding assay and vitamins A, E, C and β- carotene by high performance liquid chromatography (HPLC). 2. Gather demographics (age, race and gender), social histories (history of smoking and alcohol consumption habit) from medical records of study patients. 3. Obtain histological diagnosis of PLC from the surgical pathology reports available from the UAB Department of Pathology. 4. Determine if there are significant associations among a panel of circulating concentrations of micronutrients (folate, vitamins B-12, A, E, C and β- carotene) and the risk of being diagnosed with smoking related PLC.

REVIEW OF LITERATURE Lung Cancer: The Definition 3

Lung cancer (LC) is a disease that forms as an uncontrolled growth of abnormal cells that line air passages in lung tissue and form malignant tumors ( 9,10). PLC originates in the lungs, whereas metastatic lung cancer (MLC) spreads to the lungs from other part of the body (1,11). The main histological types of LC are non small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Among all LC cases NSCLC account for approximately 85% and SCLC accounts for 15% (12,13). Magnitude of Lung Cancer problem in the United States LC kills more people worldwide than any other malignancy. Reports from the American Cancer Society (1) and National Cancer Institute (2) indicate that there were 213,380 new cases and 169,390 deaths from LC (NSCLC and SCLC combined) in the United States in 2008, and project 214,769 new cases and 157,910 deaths in 2009, respectively (1, 3, 4). The risk of developing LC is about 23 times higher in male smokers and 13 times higher in female smokers (1).

Current and former smokers

comprise a clearly defined group at risk for LC and are an appropriate group for the study of early detection and implementation of intervention strategies. With the decrease in the prevalence of smoking among many Americans, LC has become more common among former than current smokers. In a cohort study of more than 5,000 patients whose LC was diagnosed between 1997 and 2002, only 25% were current smokers and more than half (60%) were former smokers (14). From 1998 to 2002, U.S. SEER data revealed that the median age at diagnosis for LC was 70 years. Approximately 11% of those diagnosed were under age 54 and 82% were between ages of 55 and 84. Although the age-adjusted incidence for all races, men, and women was 61 per 100,000 per year, there were significant variations between the groups. The age-adjusted incidence rates were significantly higher for both African

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American (AA) men and women relative to the Caucasian American (CA) counterparts. AA and CA women also had similar age-adjusted incidence and mortality rates, respectively (15). Notably, the incidence rate declined significantly for men, and the first decrease in LC was observed in women since 2001(15). Risk Factors of Lung Cancer Smoking The worldwide production and consumption of cigarettes has continued to increase unabated. Cigarette smoking is the main risk factor for LC accounting for ~90% of all cancer deaths, increasing the risk of this disease by at least 10-fold and as much as 20-fold, depending on smoking and medical history (16). According to published reports from the World Health Organization (17), smoking is attributed to 5 million deaths annually worldwide, and if present trends continue, 10 million smokers per year are projected to die by 2025. The median delay of time between the initiation of smoking and death from LC is approximately 50 years (18). Cigarette smoking is more strongly associated with squamous, but currently, it is also increasingly associated with adenocarcinomas that are usually located in the periphery of the lung ( 19). The incidence of adenocarcinoma has increased in many industrialized countries since the 1970s, which is thought to be due to the introduction of filter-tip cigarettes and reconstructed tobacco in the 1950s (11). The risk of developing LC increases with pack-years of smoking history (the number of packs of cigarettes smoked per day multiplied by the number of years smoked) (20). There is also a dose response relationship between smoking and LC that relates to the number of cigarettes smoked, the deepness of the inhalation of cigarettes smoked, and the duration of smoking (21). A 12-year follow up study of >1 million individuals

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consisting of current smokers, non smokers and past smokers by the American Cancer Society showed that a smoker who smoked >40 cigarettes a day for 35 to 39 years has a mortality risk from LC of 19.45% compared with individuals who have a history of 1 to 9 cigarettes a day for 20 to 24 years, whose LC related mortality risk is, 1.26% (22). While the smoking rates have declined over the years, one in five Americans smokes (10). It is estimated that 20.8% of all adults >18 yrs (45.3 million people) cigarettes in the United States; 47.4% are 18 to 44 years of age, 22.8% are 45–64 years; and 10.2% are 65 years or older, respectively (23). Cigarette smoking is more common among men (23.9%) compared to women (18.0%); highest among American Indians/Alaska Natives (32.4%), followed by African Americans (23.0%), non-Hispanic whites (21.9%), Hispanics (15.2%), and Asians (10.4%) (14). In the US, regional incidence variations in LC directly reflect smoking prevalence; specifically, the lowest and the highest incidences of LC are found in Utah and Kentucky, respectively. In Alabama, the current cigarette smoking prevalence rate among adults aged >18 years is 24.8% (24).

Mechanisms of Lung Carcinogenesis Smoking and Oxidative damage Cigarette smoke contain more than 60 compounds that include nitrosamines and polycyclic aromatic hydrocarbons (25) identified as potential carcinogens (26,27,28,29). These compounds cause accumulation of 8hydroxydeoxyguanosine (8-OHdG) (30) a product of oxidative DNA damage and an established marker for oxidative stress. Lungs from cigarette smokers contain two to three fold higher 8-OHdG which leads to mutations, some of which might be induced by oxygen free radicals, resulting in inflammatory

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responses, fibrosis and tumor development (31). Urine obtained from smokers also has a four to tenfold elevation in altered nucleotides that are known to be produced by reactive oxygen species (ROS) (32). Wood and Stockley et al reported that oxidants contained in cigarette smoke irritate epithelial cells causing a release of activating cytokines that prompt the recruitment of neutrophils and because the release of cell derived oxidants and proteases. Antioxidants inhibit oxidant mediated damage to the lung, but in case of oxidative stress, there is activation of macrophages leading to production of more proteases, hyper secretion of mucus, epithelial cell apoptosis, inflammation and at the end inhibition of the action of anti-proteases (33). A well-established concept is that smoking induces a field of epithelial cell injury (34,35). These exposed airway epithelial cells then react to cigarette smoke, and therefore the relatively accessible genes in the airway epithelial cells provide malignant transformation to the rest of the cells. Many of the genes that changed in smokers returned to nonsmoker levels within several years of smoking cessation, although approximately 15% (mostly oncogenes and tumor suppressor genes) do not return to normal even 20 to 30 yr after smoking cessation, consistent with the observation that risk for developing lung cancer remains high for decades after smoking cessation (36). In addition to accumulation of 8-OHdG carcinogens from smoke in particular polycyclic aromatic hydrocarbons have been associated with shortening of telomere (repetitive regions of the DNA that form protective caps at the ends of the chromosome) length predictive of lung cancer risk (37). Cigarette Smoking and Its Effect on Micronutrients Status Lower concentrations of antioxidants found in smokers may be due to a sustained smoke-related oxidant load that depletes the anti-oxidants (38).

Many studies have

documented that cigarette smokers have lower intakes and lower blood concentrations of

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certain antioxidants and other “ cancer protective” micronutrients, e.g., vitamin C, betacarotene, and folate (39 ,40,41). In the case of vitamin E, there is some evidence for its increased turnover in smokers, but this has little to no influence on blood concentrations, and there are no differences in dietary intake between smokers and non-smokers. Nicotine in cigarettes has an influence on taste and appetite, which may influence the intake of food rich in vitamin A and E (42). Nicotine is a highly toxic alkaloid that is a ganglionic stimulant and a depressant, and many of its effects are mediated by the release of catecholamines (43). Cigarette smoke and nicotine are thought to affect attitudes towards food intake and may lead to preferences for certain food items. Researchers have reported that smokers find sweet foods, fruit, fruit juice, and brown bread less palatable than fried foods and white bread.

These food habits may explain their

differences in intake of vitamin C and beta-carotene but not vitamin E ( 44). Further, more importantly, specific micronutrients such as folate could be lower in the circulation or in the tissues exposed to cigarette smoke (e.g.; lung epithelium and buccal mucosal cells) because of its biological inactivation by chemicals in cigarette smoke (137). The key findings of the literature that focus on these micronutrients in relation to LC are discussed below. Folate Folate plays a central role in cellular metabolism through its important function as the methyl donor for DNA methylation and nucleotides for the DNA synthesis and DNA repair. Consequently, folate mediated one carbon metabolism is linked to several health outcomes including several types of cancer (45,46,47). It has been reported that low folate status is associated with DNA strand breaks, impaired DNA repair, increased mutations, and aberrant DNA methylation which can be prevented by folate supplementation (48, 49).

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DNA methylation and DNA damage are two plausible biological mechanisms that could potentially affect cancer susceptibility.

Although several studies have attempted to

understand the association between folate and cancer risk through the study of cancer risk associated biomarkers, only the study conducted by Piyathilake et al. provides a direct association between tissue folate, LC risk and global DNA methylation. This study (39) demonstrated that low tissue folate was inversely associated with global DNA methylation in LC tissue.

Folate deficiency, perhaps by the induction of DNA

hypomethylation can also contribute to genomic instability and enhanced risk for mutations. Further, global DNA hypomethylation is shown frequently to accompany over-expression of the c-myc proto-oncogene and genomic mutations in other protooncogenes and suppresser genes such as K-ras and p53 (43). Indirect evidence is also accumulating that tissues deficient in folic acid are at an increased risk for DNA damage. Chromosomal damage to human cells in vivo as a result of folate deficiency is extensive and common: chromatid breaks, thin elongated chromosomes, despiralization, and marked accumulations of mitosis in prophase have been described (50,51,52). Furthermore, folate deficient cells are arrested at S-phase, during which mutagenicity and neoplastic transformation reach a maximum following exposure to a number of mutagens and/or carcinogens (53, 54, 55,56) Chromosomes of human cells grown in folate deficient media exhibit recurrent constitutive fragile sites, many of which are located at or near the sites of breakpoints associated with cancers ( 57). Another indicator of chromosomal integrity is the telomere length which has recently been shown in peripheral blood lymphocytes to be inversely associated with circulating folate status (58). Shortening of telomere length has also been linked to risk of LC (59). In a study comparing chromosome fragility in whole-blood cultures from smokers and nonsmokers,

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Chen et al., found that chromosome aberrations to be highly inversely correlated with red blood cell folate levels (60). In regression analysis, they found that virtually all the effect of cigarette smoking on chromosomal aberrations was explained by variations in red cell folate. Several investigators have reported that smokers have a lower concentration of folate in their plasma and red blood cells than that of non smokers ( 61,62,63,64). Repeated exposure to cigarette smoke may result in depletion of folate in respiratory epithelium of smokers thus leading to increased susceptibility to carcinogenic events (63). Chemical components of cigarette smoke such as nitrites, polycyclic aromatic hydrocarbon, cyanates and isocyanates bind folate making it biologically inactive and unavailable (62). Alcohol is another antagonist of folate as drinking alcoholic beverages greatly magnifies the cancer risk of a low-folate diet (65). There have been no reports of clear associations between lower circulating concentrations of folate and higher risk of LC. An association between low mortality from lung cancer and high intake of folate has been found in Netherland cohort study (66). Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC) study found no significant association between LC incidence and serum concentrations of folate (59). No difference was observed in the serum folate concentrations between LC cases and controls in a study conducted in Turkey. However, two randomized folate intervention studies of chemoprevention of LC have shown that folate supplementation in conjunction with B12 can reverse bronchial metaplasia, the precursor of broncogenic carcinoma among smokers (67,68). Vitamin B-12

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Vitamin B12 serves as a coenzyme in one carbon metabolic pathway essential for DNA synthesis and DNA methylation. Because of the involvement in these activities, it has been hypothesized to be associated with carcinogenesis. A case-control study conducted in Turkey demonstrated that plasma concentrations of B-12 were higher among lung cancer cases compared with controls (69). However, study of Heimburger et al. (70 ) demonstrated that supplementation with vitamin B12 along with folate reduced the severity of atypia in smokers. In contrast, ATBC study group reported that higher vitamin B12 is not associated with risk of lung cancer (71).

Antioxidants Vitamin A Epidemiological data support that the intake of fruits and vegetables that are high in pro-vitamin A lowers the incidence of LC (72). The retinoid are natural and synthetic derivatives of vitamin A. Deficiency of retinoid are associated with bronchial metaplasia and subsequently increased LC development (73). Retinods exert their therapeutic and chemopreventive effects through their role as antiproliferative, differentiation inducing and proapoptotic agent (74). Several lines of evidence suggest that like retinoids rexinoid therapy may be beneficial in the treatment of patients with LC (75). However, in the EUROSCAN study, where retinyl palmitate and/or N-acetylcisteine supplementation were used, no beneficial effects on the incidence of second primary cancer and survival were observed (76). Bexarotene a novel, multitargeted synthetic rexinoid is currently 11

being investigated in the treatment of NSCLC. In early trials, bexarotene demonstrated both satisfactory safety and promising efficacy (77,78) but the phase III trial did not increase survival in patients with advanced LC (79). Vitamin E Vitamin E consists of 4 tocopherol isomers (α, β, γ, δ) that function as a lipophilic antioxidant preventing peroxidation of lipids (80) and inhibiting inflammatory responses (81).

Their anti-inflammatory function is primarily through the inhibition of the

cyclooxygenase activities (82) which are potentially relevant in LC prevention (83). Vitamin E is also one of the most potent natural scavengers for ROS (reactive oxygen species). There have been several case-control and cohort studies on the relationship between dietary or blood levels of tocopherols and risk of LC since 1986 (84,85,86,87). Two cohort studies found a significant inverse association between dietary intakes of vitamin E and risk of LC (85, 86). Consistently, case-control studies have reported lower serum concentration of α-tocopherol among LC patients than those of matched controls (83, 84). Vitamin C or ascorbic acid Ascorbic acid plays a major role in free-radical scavenging, protection against lipid peroxidation (88) and sparing or reconstituting the active form of vitamin E (89). Several functions of vitamin C in the immune system has been described (90,91), including enhancement of leukocyte chemotaxis (92) and stimulation of interferon production. Its role in collagen synthesis and basement membrane integrity and in hyaluronidase inhibition may be an important role in the inhibition of tumor spread and micro metastases (93). Ascorbic acid is used in the treatment for vitamin C deficiency, due to improper diet, poor absorption or cigarette smoking (94). Vitamin C supplementation decreases oxidative stress biomarker F2-Isoprostanes in plasma of nonsmokers exposed 12

to environmental tobacco smoke (95). Piyathilake et al have shown that global DNA hypomethylation in lung squamous cell carcinoma tissues is associated with increased vitamin C concentrations, relative to matched uninvolved control tissues (96). Supplementation with vitamin C was not associated with a decreased risk of lung cancer (97). A pooled analysis of the data from 8 prospective studies from North America and Europe do not support that higher intakes of vitamin C reduces LC risk (98).

Beta- carotene A considerable number of epidemiologic studies conducted in the 1970s and 1980s indicated an inverse relationship between estimated intakes of beta-carotene (BC) and the risk of developing various types of cancer, especially LC (99,100). Subsequently several observational cohort and case-control studies that measured carotenoids in blood and tissues showed a consistent inverse association between circulating concentrations of BC and reduced risk of LC (41).

Mechanisms that have been proposed for such effects

include: 1) induction of cytochrome P450 xenobiotic detoxifying enzymes, 2) enhancement of gap-junctional communication, and 3) metabolism to retinoic acid that could exert biologic effects by activating nuclear retinoic acid receptors (RARs) (101,102). Later, several large-scale intervention trial were designed to test the ability of BC alone or combined with α-tocopherol or retinyl palmitate to prevent LC (103,104,105,106,107). These studies, however, failed to demonstrate protective effects of beta carotene on lung cancer. Furthermore, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study conducted in Finland (47, 48) and the beta-Carotene and Retinol Efficacy Trial study conducted in 13

the United States (49, 50) demonstrated a higher incidence of LC in current smokers, alcohol drinkers, and individuals exposed to asbestos who received BC. In contrast, exsmokers who were not exposed to asbestos showed no increased risk upon

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supplementation with BC

Several mechanism has been reported to explain these results such as (i) changes in cell oxidative status, that causes the beta-carotene antioxidant- prooxidant balance toward a prooxidant status; (ii) modulation of the levels of key proteins involved in the regulation of cell proliferation and apoptosis; (iii) reduction of retinoic acid signal pathway which down-regulates the RAR-β expression and up-regulates AP-1; (iv) interference with absorption of other cancer protective nutrients ; (v) formation of specific carotenoid oxidation products (108). Animal models have demonstrated that in the lung, the relatively high partial pressure of oxygen combined with reactive oxygen species (ROS) from tobacco smoke condensate may be conductive for BC oxidative breakdown products and act as a stimulator in the lung of cigarette smokers for free radical formation, which could be responsible for the pro-carcinogenic effects of highdose of BC supplementation (109)

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METHODOLOGY Study Population The study population consisted of 188 cases and 34 non-cases recruited between 1999 and 2001 from Dr. Robert Cerfolio’s clinic visit, Cardiothoracic Surgery Division, Department of Surgery, at The University of Alabama at Birmingham (UAB). Cases included subjects newly diagnosed and untreated with histologically confirmed smoking related PLC. Non-cases were smokers with no previous history of LC who were referred to undergo bronchoscopy and biopsy for suspected LC and were found to be free of LC. The demographic information such as age, gender and race were obtained from the medical records of the study patients. The histological diagnoses were obtained from the surgical pathology reports. The study protocol was approved by the UAB – Institutional Review Board (IRB) protocol number (X 030326005: Vitamin deficiency associated genetic/epigenetic differences and risk of lung cancer). Informed consent was obtained from each participant of the study prior to collecting data and samples. Specimen and Data Collection 15

A 10 ml fasting blood sample was collected from both cases and controls in the morning on the day of surgery by using an EDTA containing evacuated tubes. The blood samples were processed within 6 hours of collection and stored in aliquots at -800 C until analyzed for micronutrients (folate, vitamin A, B-12, beta-carotene C and E). Information concerning all confounding variables such as health habits that include smoking history and alcohol consumption were obtained from patient’s medical records. Pack years of smoking (i.e., number of packs of cigarette smokes per day time’s number of years of smoking) was calculated from the smoking history data. Laboratory Analysis Circulating concentrations of micronutrients were assayed in Dr. Piyathialke’s laboratory using standardized protocols as described below: Microbiological assay for total folate in red blood cells and in plasma General Principles. Folates (both plasma and red blood cell) were quantitated by the Lactobacillus casei (L.casei) microbiological assay. Folate in the plasma is almost exclusively in the monoglutamate form. Since L.casei responds approximately equally to all known 1-C substituted or unsubstituted, reduced or oxidized folates bearing three or less glutamyl residues in the γ- glutamyl chain, it is the best organism for the determination of the total folate levels. The basis of estimation of potency is the relationship between applied dose of growth substance (folate) and the resulting growth of the test microorganism (L.casei) in an incomplete medium (i.e., a medium containing all except one of the ingredients essential for growth). The missing ingredient is folate, which is to be assayed and which is supplied to the medium as graded doses of standard or sample.

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The growth of the microorganism results in turbidity of the initially clear test preparation. Two parameters of the growth can be measured, namely the increase in the mass of population and the increase in cell number. The most convenient and simplest method for determining the mass of the cell is based on the fact that small particles, such as bacteria, scatter light which is passed through the cell suspension. The amount of light scattering is proportional to the mass of cells present, and, because the bacterial cells are relatively small in size (mass), the number of the cells can be measured from the light that reaches a sensing device (spectrophotometer) after passing through the cell suspension. After appropriate treatment of the samples, we used a 96 –well plate adaptation of microbiological assay to quantitate total folates in our samples. Ideally, the response in growth promoting assays is directly proportional to dose. However, in practice-this ideal is not often achieved. In the case of the folate microbiological assay, the growth response is not directly proportional to dose and shows varying degrees of curvature. To avoid the necessity to interpolate from a non ideal curve, a curve straightening procedure was followed, as suggested and shown to be valid by Tsuji et al. (1967). A software program which included these features was obtained from UAB Nutrition Sciences Laboratory and was used to calculate the amount of folates in our samples. Reagents. L.casei medium were obtained from Difco Laboratories, Detroit, MI 48201. Lyophilized cultures of the assay microorganism, Lactobacillus casei (ATCC 7469a) were obtained from the American Type Culture Collection, Rockville, MD 20852. L-ascorbic acid (salt and acid) were obtained from Sigma Chemical Company, St Louis, MO 63178. Potassium phosphate (monobasic and dibasic) and glycerol were obtained from Fisher Scientific Co., Pittsburg, PA 15219. Commercially available sterile ,

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disposable plastic ware, including pipettes, pipette tips, flat-bottomed 96 –well microplates, reagents reservoirs and 0.22μm Filter/Storage system (Corning Glass , Corning, NY ),were used.

Preparation of L. casei. Freeze –dried cultures of L. casei were suspended in maintenance medium and incubated at 37º C for 24 hours. After demonstrating that the microorganism provides an adequate folate response curve, cryoprotected assay organisms were prepared according to the method described by Wilson and Horne (1982). Briefly, 50 ml of the maintenance, medium, in which the assay organism had been incubated for 18 to 22 hours, was mixed with 50 ml of sterile 80% glycerol. 500 µl aliquots of this mixture were stored in sterile tubes at -20 º C to be used as inoculate on the day of the assay, 5µl per 10 ml of assay medium. The cryoprotected inoculums were stored at -20 ºC for up to 6 months. Preparation of L. casei medium and reagents. Medium from Difco laboratories was reconstructed according to the manufacturer’s instructions and was filter sterilized using a 0.45 µm filter. Double strength Difco medium could be stored up to 4 months at 4 ºC. Sterile distilled water containing 1 mg/ml of ascorbic acid was prepared on the assay date and filter sterilized using a 0.22 µm filter. Preparation of folate Standards. All steps involving the preparation of standards were done under reduced lighting to prevent possible destruction of folate. We dissolved 100mg of folate standard in 50 ml of deionized water by adding 5 ml of 0.1 N NaOH. The pH was then adjusted to 6.8-7.2 with 0.1 N HCL. After filtration, the concentration of 5-CHO-H4 folate was determined spectrophotometrically by measuring the absorbance

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at 282 nm and using a molar extinction coefficient of 28,000. 500µl aliquots of this standard solution could be stored in sterile tubes at -70º C for up to 12 months.

Sample preparation for RBC folate assay. The conversion of red blood cell folate polyglutamates to monoglutamates was achieved enzymatically by plasma folate conjugase after incubating the hemolysate prepared (by mixing 25 µl of whole blood with 725µl of 1% ascorbic acid) at 37 º C for 20 minutes. This procedure helps to maintain the folate in its reduced state and also accomplish the first step of sample preparation for the analysis (i.e., 30 fold dilution). Enzyme treated hemolysate could be stored at -80º C for further analysis. 96- Well plate folate microbiological assay. The 96 –well plate adaptation of L.casei microbiological assay was used to measure the total folate levels in the appropriately treated samples of plasma and red blood cells (Tamura 1990; Scott et. al., 1974). Briefly, 40 µl of the samples and folate standard (7.5ng/ml) were used in the assay. The volumes of the samples and of the standard were adjusted to 300 µl using 0.1 M sterile water/1% ascorbate containing 10 mg /ml of ascorbic acid. Eight serial dilutions of sample and standard (folate standards in the range of 0.0005 to 0.15ng /0.3 ml assay well after serial dilutions) were made using a 12–channel pipette. Then 150 µl of the L.casei medium (L. casei at a concentration of 5 µl/10 ml of medium) was pipetted into all wells and incubated for 18 hours at 37º C. After incubation, the contents of each well were suspended by repeated aspiration and flushing several times with a 12 channel pipette. Bacterial growth was measured by reading the optical density at 655 nm. Plasma vitamin B-12 assay by SimulTRAC-SNB Radio assay kit

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General Principles. The basic concept of vitamin B-12 assay was using competitive protein binding assay. In competitive protein binding, the binder should have an equal affinity for the standard and the substance which is present in the patient’s sample. The unlabeled vitamin B12 competes with its labeled species for the limited number of available binding sites on its specific binder, thus reducing the amount of labeled vitamin B12 bound. Therefore, the level of radioactivity bound is inversely related to the concentration in the patient’s sample or standard. Reagents A. SimulTRAC-SNB Dithiothreitol Solution: Contains Dithiothreitol in phosphate buffer with stabilizer. B. SimulTRAC-SNB Vitamin B12 tracer: A bottle contains < 1.5 µCi Vitamin B12 in borated buffer with human serum albumin, dextran, potassium cyanide, endogenous binder blocker, dye and preservative. C. SimulTRAC-SNB Blank Reagent: Contains solid support without binder, formulated at the same solid phase concentration as the binder, in borate buffer with sodium chloride, dye and preservative. D. SimulTRAC-SNB Vitamin B12 Standards A-F: Vitamin B12 in borate buffer with human serum albumin, sodium chloride, stabilizer and preservatives. E. Extarcting Reagent: Sodium hydroxide with organic extracting enhancer and yellow dye. Equipments . Polypropylene (12X 75mm) disposable, test tube rack, semi-automatic pipette with disposable tips of delivering 100µl, 200µl and 1.0ml, vortex mixer, centrifuged with RCF 1000 x g, gamma counter for measuring [57Co] .

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Sample Preparation. Sixteen tubes were labeled for standards, beginning with 17; two tubes were numbered for each clinical sample. Working tracer 200 µl was added to all tubes and vortexed. All were incubated for 15 minutes at room temperature. 100 µl of extraction reagent was added to tubes 3-16 and all sample tubes and vortexed. The tubes were then incubated for 10 minutes at room temperature. After thoroughly mixing the blank reagent, 1000 µl of blank reagent was added to tubes 3 and 4. Then binder reagent was mixed vigorously, 1000 µl of binder reagent was added to tubes 5-16 and all sample tubes. After vortex tubes 3-16 and all samples were incubated in room temperature for 60 minutes from the last addition of the binder. Racks with tubes were covered with aluminum foil to exclude light. All of the standard and samples were centrifuged at 1000 X g for 10 minutes. The supernatant was gently decanted and discarded; even the last drop was removed by touching absorbent paper. Radioactivity was counted with gamma counter between 10,000 and 25,000 per minute. Plasma Vitamin Assays by High Performance Liquid Chromatography Method General Principles. Plasma levels of Vitamin A, C and E and of β-carotene can be measured by chemical methods or by high performance liquid chromatography (HPLC) methods. In general, chemical methods used are time consuming, cumbersome and often inaccurate due to the presence of interfering substances. High performance liquid chromatography is a technique that can be used to separate the components of a chemical mixture. These components or the solutes are first dissolved in a liquid solvent and then forced to flow through a chromatographic column under high pressure. On the column, the mixture is resolved into its components. The amount of resolution is dependent upon the extent of interaction between the solute components and the stationary phase. The stationary phase is defined as the immobile packing material in the

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column. The moving part of the HPLC system is the mobile phase, which is a liquid the interaction of the solute with mobile and stationary phases can be manipulated through different choices of the solvents and columns. Simultaneous Determination of Vitamin A and E, and of β- carotene in Plasma Reagents. Vitamin A (all Trans) and β-carotene were obtained from Sigma Chemical Co., St. Louis, MO. Vitamin E (dl-alpha tocopherol) and tocol were obtained from Hoffmann and Roche Inc., Nutley, NJ 07110. Methanol, acetonitrile, and methylene chloride were obtained from Mallinckrodt Specialty Chemical Co.; Paris, KY 40361, Hexane was obtained from EM Science, Cherry Hill, NJ 08027. Equipment. The HPLC system for simultaneous determination of vitamins A and E and of β- carotene included 150 X 3.9 mm Nova-pak C 18 (4 microns) column with a guard pack pre-column (both from Waters, Milford, MA), Waters 2487 multi-wavelength detector, Hitachi L-5000 LC and D 2000 chromatointegrator processor, Hitachi 655-61 processor, Hitachi 655A-11 liquid chromatography, and Bio-Rad auto sampler as -100. The mobile phase consisted of methanol/acetonitrile /methylene chloride (50:45:5, v/v/v) run at 1ml/min. Vitamin A (all trans retinol) was obtained from Sigma Chemical Co., St. Louis, MO, and vitamin E (dl-alpha tocopherol) and tocol were obtained from HoffmannLa Roche Inc., Nutley, NJ. Tocol is a tocopherol derivative that is used as an internal standard to correct for any loss in retinol and tocopherol during the extraction procedure. It was chosen as an internal standard because it is well separated from retinol under the normal conditions. Preparation of the standards. In preparation of the standards vitamins A and E were dissolved in the ethanol and concentrations were measured at 325 nm and 292 nm respectively using a Shimadzu UV 250 PC spectrophotometer with 1 cm light path. Tocol

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was dissolved in the ethanol (0.3 µg/ml). Coefficient of extinction: in ethanol: vitamin E= 292 nm/ε =3260, vitamin A= 325 nm/ ε =52,480. All procedures were performed in subdued yellow light. Fresh standards were prepared for each assay and standard curves were constructed by plotting peak heights against the concentrations of vitamin standards. Plasma samples from study participants were thawed and 200 μl of each placed in a separate test tube, 100 μl of the internal standard (tocol) and 100 μl ethanol for protein precipitation were added and the tubes were vortexed for 2 minutes. For extraction, 1 ml of hexane (EM Science, Cherry Hill, NJ) was added and the mixture was vortexed for 5 minute and centrifuged at 8000 RPM for 10 minutes. The top most layer hexane was carefully removed with a Pasteur pipette into another microcentrifuge tube and dried using a rotary speed-vac concentrator/evaporator (Savant Instrument Inc, Farmingdale, NY) heated to 37ºC for 25 minutes. The residue was dissolved in 200µl mobile phase and vortexes for 30 seconds. Twenty microliters of this extract was injected for chromatography. Tocol internal standard was used to determine the percent recovery in samples. For quality control, pooled normal human plasma samples were divided into two portions of high and low concentration for vitamin A and E and prepared for analysis in the same manner as the patient samples. These were run in each assay. Evaluation of the laboratory performance was assessed by comparing the results of the quality control samples with the mean and standard deviations (SD) calculated from the results of several runs of the assay. The run was rejected if any value fell outside the range of ± 2 SD from the mean.

Determination of Vitamin C/Ascorbic acid in Plasma

23

Reagents. L-ascorbic acid was obtained from Aldrich Chemical Co., Inc., Milwaukee, WI 53233. Sodium EDTA and sodium acetate were obtained from JT Baker Chemical Co., St. Louis, MO 63178. Glacial acetic acid and cysteine were obtained from Sigma Chemical Co., St. Louis, MO 63178. Q12 (dodecyltriethyl ammonium phosphate) was obtained from Regis Chemical Co., Morton Grove, IL 60053. Methanol was obtained from Mallinckrodt Specialty Co., Paris, KY 40361. Materials. The HPLC system (paired-ion reverse phase) for determination of vitamin C in plasma included Beckman Ultrasphere PTH C18 (5 micron, 250 x 4.6 mm) column with an Altex adsorbosphere guard column, LC-4B amperometric detector, Hitachi L-5000 LC controller, Hitachi 655-11 liquid chromatography pump, Hitachi D2000 chromato-integrator, and Bio-Rad AS-48 autosampler. The mobile phase consisted of 40mM EDTA, 1.5mM Q12, and 7.5% methanol adjusted to pH 4.75 with glacial acetic acid. The mobile phase was vacuum filtered through a0.2 micron nylon filter prior to use. Mobile phase was run at a flow rate of 1ml/min. Sample preparation. Blood samples were drawn into EDTA treated vacutainer tubes and the cells removed by centrifugation. Within 6 hours of sample collection, plasma was mixed with an equal volume of freshly prepared metaphosphoric acid solution (10% w/v) to precipitate proteins and to prevent oxidation of ascorbic acid. Samples were stored at -80° until analyzed. Standards. A stock solution of Vitamin C (0.5 µg/ml) was diluted in cysteine mixture (1.04 mM cysteine with 0.45 mM sodium EDTA) to make working standards ranging from 0.05 µg/ml to 0.2 µg/ml. The standard curve was constructed from peak height vs concentration. The high performance liquid chromatography (HPLC) procedure developed by Kutnink et al. (1985) was modified to measure vitamin C in

24

plasma. All procedures were performed in subdued light. Nine hundred and fifty micro liters of cysteine mixture was added to 50 µl of sample. The contents were vortexed for 1 minute and centrifuged at 8000 RPM for 5 minutes. Using plastic disposable pipettes, the supernatant was transferred into vials, capped, and injected in HPLC. Two pooled samples (high and low) and three standards were run in every 10 patient’s samples to ensure reproducibility. Statistical Method Standard statistical techniques used to analyze case-control studies were utilized to analyze the data of the study. Descriptive statistics were used to characterize the study population. The differences in the demographics such as gender and ethnicity between the cases and non-cases were determined using Chi-square tests. The difference in the median circulating concentrations of micronutrients between cases and non-cases were determined using a Kruskal-Wallis test.

The associations between circulating

concentrations of micronutrients and risk of being diagnosed with smoking related PLC were evaluated using unconditional logistic regression analysis. In these analyses the dependent variable was LC status (yes/no) and the independent predictors of primary interest were plasma folate or RBC folate, B-12, vitamin A, E, C and beta-carotene. Separate models were created for Plasma and RBC folate due to a strong positive Spearman correlation coefficient (r=0.54) between these two variables. The models were adjusted for known demographics namely, age, gender (female vs. male), ethnicity (African American vs. Caucasian American), pack years smoking history (tertiles < 30, ≥30-<60, ≥60) and alcohol consumption (yes vs. no). Circulating concentrations of micronutrients based on the tertile values for the study population (cases and non-cases combined) were used in the analysis. We compared the concentrations in the lowest

25

tertile with the middle tertile and the middle tertile with the highest tertile to determine the risk of LC for one category increase of the circulating concentrations of micronutrients. We evaluated the strength of each association by estimating the odds ratio (OR) and its 95% confidence interval (CI), and its statistical significance using Wald’s χ

2

test statistics of the null hypothesis that the OR of 1 for each variable. All

statistical analysis were considered significant at p <0.05. All analyses were performed with SAS version 9.1.3 (SAS Institute, Cary, NC, USA).

RESULTS The cross sectional analysis included 222 smokers (188 cases and 34 non-cases, Table 1). The median age of the cases (66 years) were higher compared to non cases (55 26

years, p = <0.0001).

In terms of self–reported ethnicity, a greater percentage of

Caucasian American were cases (76%) compared to non-cases (14%, p =0.3). More than half (65%) the study populations were men while 35% of the study populations were women. The distribution of the study population revealed that a greater proportion of men (55%) were more likely to be diagnosed with PLC compared to 30 % of women, but these differences were statistically non-significant (p = 0.75). With regard to smoking history, cases were more likely to have higher pack-year smoking history compared to the non-cases. No difference was observed in alcohol consumption between cases and noncases. The median circulating concentrations of plasma folate were higher among non-cases (14.35 ng/ml) compared to cases (12.48 ng/ml) although the difference was not statistically significant.

The median circulating concentrations of RBC folate were

significantly higher among non-cases (809.71ng/ml) compared to cases (714 ng/ml, p =0.001). Significant difference was also observed in the circulating concentrations of vitamin A between cases (33.9µg %) and non cases (43.6 µg %) (p =0.002). There were no significant differences between the case and non-case groups with regard to the median circulating concentration of other micronutrients (vitamin B-12, beta carotene, C and E).

27

Table 1: Demographic, lifestyle factors and circulating concentrations of micronutrients of the study population Risk factors Cases Total (N=222) 188 Median Age in years 66 Race: African American 20 (9%) Caucasian 168 (76%) Gender: Female 66 (30%) Male 122 (55%) Median Pack per year 45.5 Alcohol consumption Yes 69 (41%) No 119 (54%) Median micronutrient Concentrations Plasma Folate (ng/mL) 12.48 RBC Folate ( ng/mL) 714.85 Vitamin A (mg/mL) 43.60 Vitamin B 12 (pg/mL) 401 Beta carotene (mg%) 8.48 Vitamin C (mg%) 0.94 Vitamin E ( mg%) 0.84

Non Cases 34 55 2 (1%) 32 (14%) 11 (5%) 23 (10%) 30 16 (7%) 18 (8%)

Pvalue

14.35 809.71 33.92 326 7.39 0.59 0.93

0.260 0.680 0.002 0.710 0.260 0.450 0.190

<0.0001 0.390 0.750 0.010 0.250

The unconditional logistic regression analyses tested the association between circulating concentrations of micronutrients and risk of being diagnosed with PLC after adjusting for age, gender, race, pack year smoking history and alcohol consumption. As mentioned earlier, due to the positive correlation between plasma folate and RBC folate, the association between plasma folate and RBC folate and PLC status were tested separately. In the model that included plasma folate, we observed that increasing tertiles of circulating concentrations of folate was associated with significantly lower likelihood of being diagnosed with smoking related PLC (OR=0.45, 95% CI 0.24-0.82, p =0.009). We also observed a positive association between circulating concentrations of vitamin C and risk of being diagnosed with PLC but it only reached statistical significance (p=0.06). No significant associations were observed between circulating concentrations of vitamin 28

B-12, A, E, C and beta carotene. Details of the logistic regression model are shown in Table 2. Table: 2 Relationship between micronutrients, demographic and lifestyle factors and the risk of being diagnosed with smoking related PLC (Plasma folate in the model)

Risk Factors

OR (95% CI)

PValue

Age at enrollment (years)

1.09 (1.04-1.14)

<0.0001

African American vs. Caucasian American

2.69 (0.47- 15.21)

0.26

Female Vs Male

2.04 (0.75-5.55)

0.16

Pack per year < 30, ≥30-<60, ≥60

1.37 (0.77-2.45)

0.23

Alcohol consumption Yes vs No Circulating concentrations of micronutrients in tertiles

0.70 (0.3-1.67)

0.42

Plasma Folate (ng/mL) , <9.35, ≥9.35-<17.62, ≥17.62

0.45 (0.24-0.82)

0.009

Vitamin A (mg/mL ) ,< 32.4, ≥32.4-<49.7, ≥ 49.7

1.16 (0.6742-2.01)

0.53

Vitamin B 12 (pg/ml) < 308,≥308-<488, ≥488

1.14 (0.67-1.94)

0.62

Beta-carotene (mg%) <5.11, ≥5.11-<11.02, ≥ 11.02

1.26 (0.71-2.24)

0.42

Vitamin C tertiles (mg%) < 0.63, ≥0.63-<1.34 ≥ 1.34 Vitamin E tertiles (mg%) <0.57, ≥0.57-<1.03 ≥1.03

1.73 (0.96-3.14) 0.93 (0.54-1.60)

0.06 0.80

When plasma folate was replaced with RBC folate, we observed a similar association between RBC folate and PLC status. Increasing tertiles of RBC folate was associated with significantly lower likelihood of being diagnosed with smoking related PLC (OR=0.52, 95% CI 0.29-0.91, p =0.02). No significant associations were observed between circulating concentrations of vitamin B-12, A, E, C and beta- carotene. Details of the logistic regression model are shown in Table 3. 29

In both models we have observed that increasing age was associated with 9% increase in the odds of being diagnosed with PLC. No significant associations were observed with either pack year smoking or alcohol consumption. Table: 3 Relationship between micronutrients, demographic and lifestyle factors and the risk of being diagnosed with smoking related PLC (RBC folate in the model)

Risk Factors Age at enrollment (years) African American vs. Caucasian American

OR (95% CI) 1.08 (1.02-1.11) 2.66 (0.45- 15.74)

PValue <0.0001 0.27

Female Vs Male

1.73 (0.66-4.25)

0.26

Pack year smoking < 30, ≥30-<60, ≥60

1.62 (0.9-2.93)

0.10

Alcohol consumption Yes vs No Circulating concentrations of micronutrients in tertiles

0.67 (0.28-1.61)

0.37

RBC Folate (ng/ml) <587.5, ≥587.5-<920, ≥920

0.52 (0.29-0.91)

0.02

Vitamin A ( mg/mL) < 32.4, ≥32.4-<49.7,≥32.4 Vitamin B 12 ( pg./ml) < 308,≥308-<488, ≥488 Beta-carotene (mg%) <5.11, ≥5.11-<11.02, ≥ 11.02

1.18 (0.68-2.05) 1.12 (0.65-1.94) 1.49 (0.82-2.72)

0.54 0.67 0.18

Vitamin C tertiles (mg%) < 0.63, ≥0.63-<1.34 ≥ 1.34 Vitamin E tertiles (mg%) <0.57, ≥0.57-<1.03 ≥1.03

1.37 (0.77-2.43) 0.91 (0.53-1.57)

0.18 0.74

DISCUSSION 30

This study is one of the few studies to investigate the association between circulating concentrations of a panel of micronutrients (folate, vitamin B-12, A, E, C and Beta carotene) and risk of being diagnosed with PLC among smokers. To the best of our knowledge, this is the first study to observe a strong independent inverse association between folate (both plasma and RBC) and risk of being diagnosed with PLC after adjusting for known risk factors for lung cancer including age, race, gender, degree of smoking and consumption of alcohol.

This is also the first study to analyze the

relationship between RBC folate, an indicator of long term folate status and diagnosis of PLC.

Although there have been several epidemiological studies examining the

relationship between folate intake and LC risk, very few studies have examined the association between circulating concentrations of folate and LC risk. A case-control study conducted among former smokers demonstrated an inverse association between dietary intake of folate and risk of LC which was stronger among alcohol drinkers (110). A nested case-control study of the Japan Collaborative Cohort (JACC) that investigated the relation between serum concentrations of carotenoids, retinol, tocopherols, and folate and risk for LC cancer and death reported no significant association between circulating folate concentrations and risk of LC among its population (111). Similarly, another nested case-control study conducted within the Alpha-tocopherol, beta-carotene Cancer Prevention Cohort did not observe any significant association between serum concentrations of folate and LC risk among male smokers (112). No difference was observed in the serum folate concentrations between lung cancer cases and controls in a study conducted in Turkey (69). The design and conduct of these studies, however, were different from our study in many aspects and these differences may explain the disagreement in results between the studies.

31

Our findings of higher folate concentrations reducing the risk of PLC are interesting in the light of folate’s crucial role in the one carbon metabolic pathway. The biological mechanisms by which higher folate reduce the cancer risk are highly plausible and include folate’s effects on DNA synthesis, damage or repair and methylation of DNA and histones. Low circulating concentrations of folate in combination with carcinogen like tobacco may act synergistically to increase the malignant transformation of lung tissue through increased DNA damage and impaired DNA repair ( 113).

Previous

observations have reported that smokers have lower plasma and red blood cell (RBC) folate concentration than non smoker (114,115,116, 59).

Furthermore, smokers with

bronchial metaplasia -a pre neoplastic condition that can progress to bronchiogenic carcinoma, have been reported to have lower plasma and RBC folate concentration than smokers without metaplasia (117). This observation suggests that repeated cigarette smoke exposure causes depletion of folate in the respiratory epithelium, that makes the cells more susceptible to malignant transformation (118). It has been reported, that chemical carcinogen of cigarette smoke like nitrites, cyanates, and isocynates converts some forms of folate into biologically inactive compounds (119, damage.

120

) that makes more prone to DNA

Relton at al. suggested that, cigarette smoking is a significant source of

oxidative stress and may alter the ability of the cell to metabolize and store folate (121). It has been reported that poor dietary habits of smokers, along with associated alcohol consumption, could increase the risk for folate deficiency in smokers (21). In our study however we did not observe a significant independent association between alcohol consumption and LC risk. We did not observe significant independent associations between other “cancer protective” micronutrients such as vitamin B12, A, E, C, and beta-carotene and risk of

32

being diagnosed with PLC. Similar observations have been documented for circulating concentrations of vitamin A (122,123), beta- carotene (124,125) vitamin B-12 (126) and E (127,128,129). The strengths of the study include selection of the study population at the time of diagnosis or prior to treatment, implementation of standard procedures in the collection of fasting blood samples, use of validated and established techniques in the measurement of micronutrients and performance of micronutrient assays in blood samples within three months of storage at -80°C. Even though unequal sample size between the cases and controls could be a limitation of the study, the sample size provides 80% power to detect an odds ratio of 2.2 assuming the probability of diagnosis of LC for a reference group =0.5 . Confirmation of these results in a larger study however will increase the scientific credibility of these results.

In addition, further studies are needed to evaluate the

associations between folate and survival from PLC. Even though the mechanisms by which folate alters PLC risk such as DNA methylation and DNA damage are highly plausible, future studies are needed to confirm these mechanisms.

33

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