Nitrate, nitrite and nitrosamine: contents and analyses in selected foods; effect of vitamin C supplementation on N-nitrosodimethylamine formation in humans; and an investigation of natural alternatives to nitrites as preservatives in cured meat products by James Hsu, M. Sc Supervisor: Dr. Jayashree Arcot Co-supervisors: Dr. N. Alice Lee and Dr. Julian Cox A Thesis Submitted to the School of Chemical Sciences and Engineering In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of New South Wales October 2009 Acknowledgement Research requires a thirst for knowledge and the determination and dedication to match. In addition, a network of people including supervisors, family, friends, colleagues and laboratory technicians all works as a team to give advice and support when need it. That being said, I would like to give my gratitude to my main supervisor Dr. Jayashree Arcot for her consistent and helpful supervision throughout my PhD candidature. I would also like to extend my gratitude to Dr. Alice Lee and Associate Professor Dr. Julian Cox for their expertise in specific parts of my research. Big thanks goes to my close friends and colleagues for lending an ear or a shoulder whatever the occasion calls for, or simply by hanging out with me to save my sanity at times. The laboratory staffs are the glue that holds everything together, without their experience and knowledge the lives of any PhD candidates would be made a lot harder. Since I started my PhD in 2004, Eileen and Evyone had helped me in many ways, but special thanks go to Camillo for his dedication, professionalism and support that just made it all worthwhile. All this could only happen with the financial support from Bruce Veness Chandler Award, and to my mum Stephanie and my siblings Patricia and Frank for showing their love in their own distinct ways. Finally but not least, my gratitude to my late father Michael and my grandmother for their early upbringing that made me the man I am today. 2 Abstract Dietary nitrite intake has been implicated in numerous gastrointestinal cancers in humans due to the formation of a group of carcinogens called N-nitroso compounds. The need to estimate their intake is vital in establishing at risk population and to monitor and perhaps one day manage their dietary intake. This is the first study to estimate nitrate and nitrite in selected vegetables, cured and fresh meat in Australian food supply using ion-paired reversed-phased HPLC. Nitrite content in meat products analysed ranged from 0 to 83.9 mg/kg in medallion beef and Frankfurt, respectively; nitrate content ranged from 18.7 mg/kg in minced beef to 142.5 mg/kg in salami. The nitrite content was below the maximum limit set by the Food Standards Australia and New Zealand. Nitrate content in selected vegetables ranged from 123 to 4850 mg/kg in Iceberg lettuce and English spinach, respectively; only minimal nitrite at 20 mg/kg was present in Gai choy, which was most likely due to bacterial contamination during storage. Based on the food consumption pattern of Australians, the dietary nitrite and nitrate intake from bacon were 1.51 and 3.42 mg per capita per day, which was below the Adequate Daily Intake set by the European Union Scientific Committee for food in 1995. Taking into considerations of oral nitrate reduction to nitrite and the endogenous nitrate formation, the upper extreme of dietary nitrite and nitrate intake in Australians were 44 and 2.4 times over the ADI, respectively. However, this does not take into effect of other dietary promoters and inhibitors. Eighteen healthy human volunteers were put on a low nitrate, nitrite and antioxidant diet for three days during which they were fed one serving of cured meat with and without 500 mg of vitamin C. Using GC- MS, N-nitrosodimethylamine was not detected in the urine before or after vitamin C supplementation, suggested that a diet low on nitrate and nitrite cannot produce NDMA and thus may reduce the risk of developing gastrointestinal cancers. Different extraction methods and combination of herbs and spices were demonstrated in vitro to show inhibition against B. cereus, Escherichia coli, Listeria monocytogenes, Salmonella Enteritidis and Staphylococcus aureus. In addition, autoclaved turmeric powder at 0.3 % (w/v), hot water extracted turmeric with ginger at 0.5 % and rosemary at 1.0 % showed growth inhibition against Clostridium sporogenes, which was used as a surrogate for Clostridium botulinum. The use of these combinations of herbs and spices may replace or at least reduce the use of nitrite as a preservative in cured meat products to prevent botulism and reduce dietary nitrite intake. 3 Table of Contents Acknowledgement ................................................................................................................. 2 Abstract ..................................................................................................................................... 3 Table of Contents .................................................................................................................... 4 List of Figures .......................................................................................................................... 8 List of Tables ......................................................................................................................... 10 List of Abbreviations .......................................................................................................... 12 Chapter 1 ................................................................................................................................ 15 Introduction .......................................................................................................................... 15 1.1 Experimental aims ................................................................................................................. 16 1.1.1 Nitrate and nitrite in Australian sourced vegetables and cured meat and their estimated dietary intake by Australians ........................................................................................ 16 1.1.2 Effects of vitamin C supplementation on N‐nitrosodimethylamine formation in healthy human volunteers ................................................................................................................... 16 1.1.3 Anti‐Clostridia properties of herbs and spices and possible substitution for sodium nitrite ............................................................................................................................................ 16 Chapter 2 ................................................................................................................................ 18 Nitrate and nitrite ............................................................................................................... 18 2.1 Dietary sources of nitrates and nitrites ......................................................................... 18 2.1.1 Plant food ......................................................................................................................................... 18 2.1.2 Cured meat ...................................................................................................................................... 20 2.1.3 Water ................................................................................................................................................. 21 2.2 Nitrate, nitrite and its derivative ...................................................................................... 22 2.2.1 Mechanisms of nitrate reduction to nitrite ........................................................................ 22 2.2.2 Mechanisms of nitrite reduction to nitric oxide ............................................................... 22 2.2.3 Metabolism of nitrate, nitrite and nitric oxide ................................................................. 23 2.2.4 Physiological roles of nitrate, nitrite and nitric oxide ................................................... 26 2.2.5 Immunological effects and disease ........................................................................................ 29 2.3 Toxicity and Metabolism ..................................................................................................... 30 2.3.1 Nitrate and nitrite ......................................................................................................................... 30 2.4 Epidemiology studies ............................................................................................................ 34 2.4.1 Gastric cancer incidence ............................................................................................................ 34 2.4.2 Gastric cancer risk factors ......................................................................................................... 38 2.4.2.1 Carbohydrate............................................................................................................................................. 39 2.4.2.2 Saturated fat .............................................................................................................................................. 39 2.4.2.3 Alcohol .......................................................................................................................................................... 40 2.4.2.4 Salt, pickled and smoked food ..........................................................................................................