COMPREHENSIVE STUDY OF THE OXIDATIVE STABILITY OF THE MOST CONSUMED ULTRA - PROCESSED FOODS IN THE USA By Lisaura Maldonado - Pereira A DISSERTATION Submitted to Michigan State University i n partial fulfillment of the requirements f or the degree of Chemical Engineering Doctor of Philosophy Biosystems Engineering Dual Major 2021 ABSTRACT COMPREHENSIVE STUDY OF THE OXIDATIVE STABILITY OF THE MOST CONSUMED ULTRA - PROCESSED FOODS IN THE USA By Lisaura Maldonado - Pereira Ultra - processed food s (UPFs) are characterized for being inexpensive, highly processed, rich in calories, but low in some essential micronutrients such as mineral s and vitamins. Nearly 60% of the calories consumed by the average American come from UPFs (Martínez Steele et al., 2016 ). Processing of food plays a crucial role in the overall chemical safety of the American diet (also known as the Western diet) since different manufacturing conditions promote the formation of unintended and harmful compounds in the final product. This is the case of cholesterol oxidation products (COPs) which have been associated with the development of several chronic diseases. COPs are derived from the oxid ation of c holesterol and can be triggered by different parameters such as light, heat, radiation , metal ions , and other agents. Until this study, there has been no database of these compounds in the Western diet and their level of exposure of the US population. A total of 63 UPFs were tested. Fatty acids, cholesterols and its oxidation products, tocopherols and phytosterols were comprehensively assessed by chromatographic means. Oxidative status of the Western diet was evaluated by the quantification of secondary oxidation products such as malondialdehyde - - - - - Epoxy, 7 - Ket o, Triol, 6 - - OH, 22 - OH, 24 - OH, and 25 - OH), and other sterols (cholesterol, phytosterols, and tocopherols) were detected. An assessment of the level of exposure of COPs was performed using the Stochastic Human Exposure and Dose Simulation (SHEDS) d eveloped by the US Environmental Protection Agency (EPA) (EPA, 2020). Forty - four percent of the samples showed a different fat content than those reported content than the reference value from their nutritional label. Twenty - six percent of fast food (FF) meals showed a high PUFA content which is a type of healthy fat that improve s cardiovascular health (Harris, 2007; Lu et al., 2011), brain function, and overall he alth during pregnancy (Koletzko et al., 2008) . Saturated fatty acid (SFA) content in UPFs was - Sitosterol was the most abundant phytosterol. However, differences in concentrations were observed depending on t he f ood matrix and ingredients added to the food item throughout its preparation . Similarly, total COP content varied among food matrices and ingredients added. This means that food matrix, n play an important role in th e distribution of these sterols. Lastly, infants (6 - 12 months) could be exposed to upwards of 309.56 mg/kg/6 mo. (0.0031 mg/kg/6 mo.) . Since there is no study addressing uld be done to determine if it should be considered a health risk . This study provides a complete overview of the oxidative lipid status of the most popular UPFs in the Western diet as well as an assessment of the exposure level of these compounds in one of the most vulnerable groups: infants . N utritional quality and dietary patterns seem to be jeopardized by prices and popularity of UPF meals , resulting in a public health issue that should be addressed. iv This dissertation is dedicated to my family and Obie . T hank you for always believ ing in me. v ACKNOWLEDGEMENT Life has mysterious ways to teach each of us the meaning of hard work , resilience, group support, perseverance , compassion, and courage . Many people have been key to the success of this personal and yet collective milestone. I would like to express my infinite gratitude to several people , starting with my mentor, advisor, and second mother, Dr. Ilce Medina - Meza . Thank you for believing in me. Your active guidance throughout the completion of my doctoral degree has been my compass in this winding adventure. You are a n exceptional role model and have helped to shape me into a better professional and academic. Also, I will always be grateful to my graduate com mittee for their willingness to help me throughout this journey. Lastly, t his triumph could not be possible without the support of Dr. Perc y Pierre . Thank you for allowing me to stay in school an d continu e my doctorate degree . Your encouragement and help during difficult times are t he base of this achievement. I am eternally thankful to both of my Departments (the Chemical Engineering Department and the Biosystem and Agricultural Department) for supporting me throughout my entire doctoral program . Finally , I extend my appreciation to those who could not be mentio ned here but have played their role to inspire, help, and continuously encourag e me to give my all in this process. Lisaura Maldonado - Pereira vi TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ......................... i x LIST OF FIGURES ................................ ................................ ................................ ........................ x KEY TO ABBREVIATIONS ................................ ................................ ................................ ............ x i C H APTER 1: IMPORTANCE OF COP S IN FOOD TOXICITY ................................ ........................... 1 1.1 INTRODUCTION ................................ ................................ ................................ ....... 1 1.2 OCCURRENCE OF COPS IN BIOLOGICAL SYSTEMS ................................ ................. 4 1.2.1 Absorption and T ransport of COPs ................................ ......................... 4 1.2.2 Biological and Pathological Activities of COPs ................................ ....... 6 1.2.3 COPs as Biomarkers of Chronic Diseases ................................ ............ 10 1.2.4 COPs as a Promoter of Cellular Apoptosis ................................ ........... 12 1.2.5 COPs and Cell Survival Mechanisms ................................ .................... 15 1.3 DIETARY COPS: OCCURRENCE IN FOOD ................................ ............................... 16 1.3.1 Food Processing Triggers COPs Formation in Foods ............................ 17 1.3.2 Baby Foods ................................ ................................ ........................... 19 1.3.3 Food Chain: Packaging and Storage ................................ .................... 20 1.4 COPS: A FOOD TOXICOLOGICAL TARGET? ................................ ............................. 22 1.4.1 Fate of the COPs: Risk and Exposure Assessment .............................. 22 1.4.2 COPs as Biomarkers of Food Safety ................................ ..................... 24 1.5 PARADIGM SHIFTS AND FUTURE CHALLENGES ................................ ................... 25 1.5.1 Oxidative Mechanisms: Holistic Approach ................................ ........... 25 1.6 CONCLUSIONS ................................ ................................ ................................ ....... 2 6 CHAPTER 2: PHYTOSTEROLS AND THEIR OXIDA TIVE PRODUCTS IN INFANT FORMULA ........ 2 8 2.1 INTRODUCTION ................................ ................................ ................................ ...... 28 2.2 PHYTOSTEROLS FOOD SOURCES ................................ ................................ .......... 30 2.3 HEALTH BENEFITS OF PHYTOSTEROLS ................................ ................................ 31 2.4 PHYTOSTEROLS OXIDATION ................................ ................................ ................. 34 2.5 POPS BIOLOGICAL TOXICITY ................................ ................................ .................. 36 2.6 INFANT FORMULATION: A CRITICAL NEED ................................ ........................... 37 2.7 PROCESSING TECHNOLOGY RELATED TO IF ................................ ........................ 41 2.8 THE CHALLENGE OF FOOD PROCESSING: LIPID OXIDATION ............................... 43 2.9 CONCLUSIONS ................................ ................................ ................................ ...... 45 CHAPTER 3: EVALUATION OF THE NUTRITIONAL QUALITY OF ULTRA - PROCESSED FOODS (READY TO EAT + FAST FOOD): FATTY ACID COMPOSITION ................................ ........ 4 7 3.1 INTRODUCTION ................................ ................................ ................................ ...... 47 3.2 MATERIALS AND METHODS ................................ ................................ .................. 49 3.2.1 Materials, Chemicals, and Reagents ................................ ..................... 49 3.2.2 Sample Collection ................................ ................................ ................... 50 3.2.3 Sample Preparation ................................ ................................ ................ 51 vii 3.2.4 Lipid Extraction ................................ ................................ ....................... 51 3.2.5 Fa t ty Acid Methyl Esters (FAME) ................................ ............................. 51 3.2.6 Statistical Analysis ................................ ................................ .................. 52 3.3 RESULTS AND DISCUSSION ................................ ................................ .................. 53 3.3.1 Total Fat, Sugar, and Sodium in UPFs ................................ .................... 53 3.3.2 Fatty Acid Profile by Food Category ................................ ........................ 53 3.3.3 Saturated Fatty Acids (SFA) ................................ ................................ .... 58 3.3.4 Monounsaturated Fatty Acids (MUFA) ................................ .................... 61 3.3.5 Polyunsaturated Fatty Acids (PUFA) ................................ ....................... 64 3.4 CONCL USIONS ................................ ................................ ................................ ....... 69 CHAPTER 4: CHOLESTEROL OXIDATION PRODUCTS ASSESMENT IN ULTRA - PROCESSED FOODS IN THE WESTERN DIET ................................ ................................ .................... 70 4.1 INTRODUCTION ................................ ................................ ................................ ...... 70 71 4.1.2 Ultra - Processed Foods and NOVA Classification ................................ .... 73 4.1.3 COPs as Bio markers ................................ ................................ ............... 76 4.1.4 Effect of Food Processing on Cholesterol Oxidation .............................. 77 4.2 MATERIALS AND METHOD ................................ ................................ ..................... 78 4.2.1 Materials, Chemicals, and Reagents ................................ ..................... 78 4.2.2 Sample Collection and Preparation ................................ ....................... 78 4.2.3 Sample Prepar ation ................................ ................................ ................ 79 4.2.4 Lipid Extraction ................................ ................................ ....................... 80 4.2.5 Thiobarbituric Acid Reactive Substances (TBARS) ................................ . 80 4.2.6 Total Cholesterol and Phytosterols Content ................................ ........... 81 4.2.7 COPs Quantification ................................ ................................ ............... 82 4.3 STATISTICAL ANALYSIS ................................ ................................ .......................... 83 4.4 PRELIMINARY RESULTS ................................ ................................ ........................ 83 ................................ ................................ ............ 84 4.4.2 Dietary Ox idized Sterols DOxS ................................ ................................ 85 4.4.3 Multivariate Analysis of DOxS ................................ ................................ . 86 4. 5 RESULTS FROM THE COPS ASSESSMENT OF UPFS ................................ ............. 87 4.5.1 Fat Content in UPFs ................................ ................................ ................ 87 4.5.2 TBARS ................................ ................................ ................................ ..... 87 4.5.3 Total Cholesterol Content ................................ ................................ ....... 88 4.5.4 Phyt o sterols and Tocopherols Quantification ................................ ........ 89 4.5.5 COPs 4 .6 DISCUSSION ................................ ................................ ................................ ........ 101 4.6.1 Fat in UPFs ................................ ................................ ........................... 101 4.6.2 Secondary Oxidation Products (TBARS) ................................ ............... 102 4.6.3 Total Cholesterol ................................ ................................ ................... 103 4.6.4 Phytosterols and Tocopherols in UPFs ................................ ................. 104 4.6.5 COPs Occurrence in the Western Diet ................................ .................. 106 4.7 CONCLUSIONS ................................ ................................ ................................ ..... 109 CHAPTER 5: LIPID OXIDATION SPECIES IN THE WESTERN DIET A HEALTH AND NUTRITION THREAT ................................ ................................ ................................ ...................... 1 10 viii 5. 1 OVERALL SIGNIFICANCE ................................ ................................ ...................... 110 5.2 LIPID OXIDATION AND FOOD SAFETY ................................ ................................ .. 111 ................................ .......... 111 5.4 LIPID OXIDATION AND FOOD LAW ................................ ................................ ....... 112 5.5 LIPID OXIDATION SPECIES AND HUMAN HEALTH ................................ ............... 113 5.5.1 Dietary Exposure Assessment as a Public Health Tool ........................ 114 5.5.2 SHEDS - HT Exposure Model ................................ ................................ .. 115 5.5.2.1 How Does SHEDS Work? ................................ ................................ ... 11 7 5.5.2.2 Population Module ................................ ................................ ............ 119 5.5.2.3 Exposure Asse ssment Test 2 nd Stage Baby Foods ........................ 119 5.6 CONCLUSIONS ................................ ................................ ................................ ..... 120 5.7 FUTURE WORK ................................ ................................ ................................ .... 121 APPENDICES ................................ ................................ ................................ ......................... 123 APPENDIX A: The R ole of C holesterol O x idation P roducts in F ood T oxicity ( J ournal P aper) ................................ ................................ ................................ ................................ ............ 1 2 4 APPENDIX B: Phytosterols and t heir O xidative P roducts in I nfant F ormula ( J ournal P aper ) ................................ ................................ ................................ ................................ ............ 15 8 APPENDIX C: List of A ll F ood M eals, T heir T est C odes and F ood G roup ............................ 20 5 APPENDIX D: Conditions of the C ollection and P reparation P rocess of R eady - to - E at (RTE ) items ................................ ................................ ................................ ................................ ... 20 8 APPENDIX E: Conditions of the C ollection and P reparation P rocess of F ast - F ood (FF) M eals ................................ ................................ ................................ ................................ ............ 2 1 1 APPENDIX F: Sterols and P hytosterols C ontent in R eady - to - E at (RTE) and F ast - F ood (FF) meals ................................ ................................ ................................ ................................ .. 21 3 LITERATURE CITED ................................ ................................ ................................ ................ 2 1 7 ix LIST OF TABLES Table 1 - 1 : Classification table of the origin (endogenous and exogenous) of the most detected COPs published in Maldonado - Pereira et al., 201 8 ................................ .................. 4 Table 1 - 2: Summary of the table reported in Maldonado - Pereira et al., 2018 which describes the relationship between COPs and different chronic diseases. Dietary COPs are highlighted ................................ ................................ ................................ ................................ .................. 7 Table 2 - 1 : Summary table of the phytosterol content in oils and foodstuff published i n Kilvington el at. 2019 ................................ ................................ ................................ ............. 32 Table 3 - 1: Food ID used in this study and unpublished results of total fat, sugar, sodium, and calories per serving found in the different UPFs ................................ ................................ .... 5 4 Table 3 - 2: Unpublished results of the saturated fatty acid composition in RTE and FF meals ................................ ................................ ................................ ................................ ................ 60 Table 3 - 3: Unpublished results of monounsaturated and polyunsaturated fatty acid composition in RTE and FF meals ................................ ................................ ........................... 6 2 Table 3 - 4: Unpublished results of the fatty acid profile of UPFs by foo category (mean and range) ................................ ................................ ................................ ................................ ...... 6 5 Table 4 - 1: Unpublished results of MDA concentration in RTE items and FF meals ............... 9 1 Table 4 - 2 : Unpublished results of total fat and cholesterol contents in RTE items and FF meals ................................ ................................ ................................ ................................ ....... 9 2 Table 4 - 3 : Unpublished results of COPs concentrations in RTE items ................................ ... 9 5 Table 4 - 4: Unpublished results of COPs concentrations in FF meals ................................ ..... 9 9 Table 5 - 1 : Unpublished results of COPs exposure in infant (6 - 12 months old) ................... 1 20 Table 6 : Processed and Ultra - processed meals ID used in this study ................................ .. 20 6 Table 7 : Detailed information for the collection and preparation process of RTE items in this analysis. ................................ ................................ ................................ ................................ 209 Table 8 : Detailed information for the collection and preparation process of RTE items in this analysis ................................ ................................ ................................ ................................ . 212 Table 9 : Unpublished results of the sterols and phytosterols content in RTE and FF meals in UPFs ................................ ................................ ................................ ................................ ...... 214 x LIST OF FIGURES Figure 1 - 1 : Diagram obtained from Maldonado - Pereira et al. 2018 which explains the different structures of the main COPs according to their site of oxidation ................................ ............. 2 Figure 2 - 1 : Structures of the most common phytosterols obtained from Kilvington et al. 2019 ................................ ................................ ................................ ................................ ....... 29 Figure 2 - 2: Figure published in Kilvington et al. 2019 describing the general phytosterol oxidation pathway where R denotes a specific phytosterol side chain ................................ .. 35 Figure 2 - 3: Table published in Kilvington et al. 2019 describing the phytosterol concentration reported in infant formula ................................ ................................ ............... 3 9 Figure 2 - 4: Infant formula milk powder manufacturing process (Kilvington et al. 2019) ...... 4 3 Figure 3 - 1: Unpublished results of (A) fat conten t vs. food categories, (B) SFA vs. food categories, (C) MUFA vs. food categories, (D) PUFA vs. food categories, (E) calories per servings vs. food categories, and (F) sodium vs. food categories ................................ .......... 5 7 Figure 4 - 1: NOVA food classification based on food processing ................................ ............ 7 5 Figure 4 - 2: Cholesterol and oxidized sterols ................................ ................................ ........... 8 5 Figure 4 - 3: Heatmap of the correlation of lipid profiles across food meals ........................... 8 6 Figure 4 - 4: Distribution of cholesterol contents between food groups ................................ 10 4 Figure 4 - 5: Distribution of phytosterols between food groups ................................ ............. 10 5 Figure 4 - 6: Distribution of COPs between food groups ................................ ......................... 10 7 Figure 5 - 1 : Study's significance in the three main areas: Public Health, Nutrition, and Food Market ................................ ................................ ................................ ................................ ... 1 10 Figure 5 - 2 : Importance of an Exposure Assessment ................................ ............................ 11 4 Figure 5 - 3 : SHEDS Input Categories (Isaacs et al., 2014 ) ................................ .................... 11 6 Figure 6 : The Role of Cholesterol Oxidation Products in Food Toxicity (Journal Paper) ....... 125 Figure 7 : Phytosterols and their Oxidative Products in Infant Formula (Journal Paper) ....... 159 xi KEY TO ABBREVIATIONS COP s cholesterol oxidation products CHOL cholesterol ROS reactive oxygen species 7 - OH 7 - hydroxycholesterol 7 - KETO 7 - ketocholesterol 5,6 - EPOXY 5 ,6 - epoxycholesterol - EPOXY - epoxycholesterol LDL low density lipoprotein HDL high density lipoprotein VLDL very low - density lipoprotein LXR liver X receptor - OH 7 - hydroxycholesterol - OH 7 - hydroxycholesterol 24S - O H 24S - hydroxycholesterol 27 - OH 2 7 - hydroxycholesterol AR androgen receptor MS multiple sclerosis 25 - OH 2 5 - hydroxycholesterol 20 - OH 2 0 - hydroxycholesterol TRIOL Cholesteane - , 5 , 6 - triol HD AD MCI mild cognitive impairment SFA saturated fatty acids MUFA monosaturated fatty acids PUFA p olyunsaturated fatty acids xii DHA docosahexaenoic acid ARA arachidonic acid FF fast food RTE read y - to - eat UPFs ultra - processed foods PFs processed foods TDS total dietary study FDA F ood and Drug Administration ACAT a cyl - coenzyme A cholesterol acetyltransferase UHT ultra - high temperature U937 Human lymphocyte cells MCF7 Human mammary gland, breast; derived from metastatic site: pleural effusion CYP Cytochrome P450 enzyme family HUVE C Human umbilical vein endothelial cell POPs Phytosterol Oxidation Products IF Infant Formula SHEDS Stochastic Human Exposure and Dos e Simulation SHEDS - HT Stochastic Human Exposure and Dose Simulation High Throughput 1 CHAPTER 1 : IMPORTANCE OF COPS IN FOOD TOXICITY T he content of this chapter has been previously published in the Food and Chemi cal Toxi cology Journal. DOI: 10.1016/j.fct.2018.05.059 1.1 INTRODUCTION Cholesterol is a key component of mammalian cells with a crucial role in different b iological processes . It is a precursor of a variety of hormones, vitamin D and bile acid production cycle (Vesa M Olkkonen, Gylling, & Ikonen, 2017) . Cholesterol is synthetized endogenously in the body in amounts necessary to sustain l ife. The pres ence of reactive oxygen species ( ROS ) due to cell oxidative s tress or exposure to radical species increases cholesterol s usceptibility to oxidation (I. G. Medina - Meza & C. Barnaba, 2013; L. L. Smith, 1996) . This non - enzymatic oxidation occurs at the C 5 - C 6 double bond via n ucleophilic attack by radicals and other reactive sp ecies. Cholesterol sidechain is also susceptible of radical attack (Iuliano, 2011; L. L. Smith, 1996) . R OS - mediated oxidation can be triggered by light (Maerker & Jones, 1993a; Ilce Gabriela Medina - Meza & Barbosa - Cánovas, 2015; Ilce Gabriela Medina - Meza, Rodriguez - Estrada, Sergio Garcia, & Lercker, 2012; Medina Meza, Rodriguez Estrada, Lercker, Barnaba, & García, 2014) , Heat (Chien, Wang, & Chen, 1998; D. Derewiaka & Molinska nee Sosinska, 2015) , Radiation (Maerker & Jones, 1993a) , metal ions (Hur, Park, & Joo, 2007b; Jo, Ahn, & Lee, 1999) and other agents that have the ability to lower the energetic r equirements for the reaction to occur (Beltran, Pla, Capellas, Yuste, & Mor - Mur, 2004; I. Medina Meza & Barnaba, 2013) . Cholesterol oxidation produces a group of molecules t hat preserve the steroidal motif of the parent co mpound, but with an 2 a dditional hydroxyl, ketone, or epoxy group (Savage, Dutta, & Rodriguez - Estrada, 2002) ( Figure 1 - 1 ). They are commonly known as cholesterol oxidation products ( COPs ), and d epending on the site of oxidation, COPs can be distinguished as - ring, - ring, or side - chain oxidation products ( Figure 1 - 1 and Table 1 - 1 ) . E ach of these molecules have d emonstrated biochemical, biological, and even biophysical activities in m ammalian cells and subcellular components (Gabriella Leonarduzzi et al., 2005) . Endogenous COP s have been considered as key intermediates in bile acid a nd steroid hormone biosynthesis or as autoxidation products in tissues, e specially under conditions of oxidative stress (Ikegami et al., 2014; Vesa M. Olkkonen, Béaslas, & Nissilä, 2012; Vaughn et al., 1997) . O n the o ther hand, exogenous COPs are mainly derived from diet by consumption of high cholesterol - containing foods. This intake results in the accumulation of COP s i n several organs and tissues, as it Figure 1 - 1 : Diagram obtained from Maldonado - Pereira et al. 2018 which explains the different structures of the main COPs according to their site of oxidation 3 has been extensively reported in previous studies (Biasi et al., 2013; Miyoshi, Iuliano, Tomono, & Ohshima, 2014; Ogino et al., 2007; Ros, 2000) . Animal f ood products including meat and meat products (K. Al - Ismail, 2002; KM Al - Ismail, Herzallah, & Humied, 2007; Huei, 2012; Grau, Codony, Grimpa, Baucells, & Guardiola, 2001; Novelli et al., 199 8; Osada, Kyoichi, Shinichi, Shingo, & Michihiro, 2000; Zubillaga M. P. and Maerker, 1991) ; (Hur, Lee, & Lee, 2015) , fish (Echarte, Conchillo, Ansorena, & Astiasarán, 2004; Hernández, 2014; Osada et al., 1993; Paola Zunin, Boggia, & Evangelisti, 2001) , eggs and egg p roducts (L. J. Chen, Lu, Chien, & Chen, 2010) , cheese (Nourooz - Zadeh, 1988) , milk (Calderón Santiago, Peralbo Molina, Priego Capote, & Luque de Castro Mar í a, 2012; Dionisi, Golay, Aeschlimann, & Fay, 1998) and o ther dairy products (Angulo, Romera, Ramirez, & Gil, 1997; Gorassini, Verardo, Fregolent, & Bortolomeazzi, 2017; Kumar, 1992; Mariutti & Bragagn olo, 2017; Nielsen, 1995; X. D. Sun & Holley, 2010) are the major source of dietary COP s (A Grandgirard, Guardiola, Dutta, Codony, & Savage, 2002; Hur, Park, & Joo, 2007a; Ogino et al., 2007) . The content of COP s in food has been largely overlooked, despite a consistent body of evidence on their biological and pathological activities in humans. COP s are unavoidable and unintentionally formed during food processing, storage, handling and even household preparations, making human exposure a tangible risk. This chapter will discuss the gap between the scientific evidence of COP s formation in biologica l systems, and the chemical exposure and toxicological significance through dietary intake. First, data regarding the occurrence of COP s in foods and other biological matrices will be reviewed . Second, the compound - specific biological and pathological sign ificance will be addressed. 4 Table 1 - 1 : Classification table of the origin (endogenous and exogenous) of the most detected COPs published in Maldonado - Pereira et al . , 2018 . Subsequently, the factors that enhance their formation in food products will be analyzed . F inally , the importance of an accurate estimat ion of these compounds through a risk exposure assessment will be highlighted . Overall, this chapter will strengthen the need of a holistic approach on cholesterol oxidatio n and COPs, an emerging field already biomarkers for food safety and toxicology. 1.2 OCCURRENCE OF COPS IN BIOLOGICAL SYSTEMS 1.2.1 Absorption and transport of COPs The fate of dietary cholesterol and cholesterol - derivatives has been studied in different animal models, including humans. Dietary cholesterol appears in the lumen of the intestine associated with triglycerides and phospholipids in lipid emulsion. These lipid 5 micelles are digested by lipases, and cholesterol is released for its transport to the brush border of the small intestine for absorption by the mucosal cells (Shoshana Rozner and Nissim, 2006) . Cholesterol is absorbed in the human small intestine thro ugh ABCG5/ABCG8 and NPC1L1 transporters, which effluxes a portion of the absorbed cholesterol back into the small intestinal lumen and drives the cholesterol uptake in the enterocytes (Shoshana Rozner and Nissim, 2006) . On the contrary, the absorption pathway of COPs has not been characterized in its completeness due to their chemical diversity, polarity, and abundance in the diet. Studies have shown that COPs are absorbed i n the small intestine at a lower rate (K. Osada, Sasaki, & Sugano, 1994) , probably due to their lower solubility in micelles. Cholesterol, fatty acids and eventually COPs are then assembled i nto large intestinal lipoproteins, called chylomicrons. Cholesterol is esterified by the acyl - coenzyme A cholesterol acetyltransferase (ACAT) before incorporation into the chylomicrons. Intragastric administration of a mixture of COPs in rats revealed that their absorption is related with changes in the composition of lymph chylomicron after 2 4h (Emanuel, Hassel, Addis, Bergmann, & Zavoral, 1991) . The authors also found a time - correlation between the incorporation of individual COPs species into the lymph chylomicrons. 7 - OH isomers were early incorporated (3 h), followed by 7 - keto and - epoxy at 4 and 5 h, respectively (Vine et al., 1997, 1998). 7 - OH isomers and 7 - keto are the COPs commonly identified in lipoproteins (Kuver, 2012) . The highest le vels of COPs are present in low - density lipoproteins (LDLs) and to a lesser extent in high - density lipoproteins (HDLs) and very - low - density lipoproteins (VLDLs) (Vaya et al., 2001; A. Vejux & G. Lizard, 2009) . Huang et al. (2015) suggested the revision of the cholesterol transport hypothesis since several epidemiological studies demonstrated the cholesterol efflux (reverse cholesterol transport) play s an important role in the absorption and removal of excess of cholester ol in plasma. 6 Notwithstanding, no information is still provided about the implication of COPs in the absorption of cholesterol. 1.2.2 Biological and pathological activities of COPs In the last decades, there has been mounting evidence of COPs exert ing biological and pathological activities in both in vitro and in vivo systems, with potential health concerns for humans (Gabriella Leonarduzzi et al., 2005) . Several studies have demonstrated that COPs can exert pro - inflammatory (Biasi et al., 2013; Lemaire - Ewing et al., 2005; Miyoshi et al., 2014; Virginio et al., 2015) , pro - oxidant (Biasi et al., 2009; Mariutti & Bragagnolo, 2017; Seet et al., 2009) , pro - fibrogenic (Gargiulo, Gamba, Testa, Leonarduzzi, & Poli, 2016) and pro - apoptotic (Colles, Maxson, Carlson, & Chisolm, 2001; A. Vejux & G. Lizard, 2009) activities in several cell lines (Raza et al., 2016; A. Vejux & G. Lizard, 2009; Zarrouk et al., 2014) . COPs have also shown specific deleterious properties such as cytotoxicity, mutagenicity (Sevanian & Peterson, 1986) , carcinogenicity (Homma et al., 2004) , Alzheimer 's disease (Marwarha & Ghribi, 20 14) , Parkinson's disease (Bjorkhem et al., 2013; C. Y . Lee, Seet, Huang, Long, & Halliwell, 2009; Leoni & Caccia, 2011) , age - onset macular dege neration (Javitt & Javitt, 2009) as well as cataracts (Girão, Mota, Ramalho, & Pereira, 1998) , osteoporosis (H. L iu, Yuan, Xu, Zhang, & Wang, 2004) , colon carcinoma (Biasi et al., 2009; Roussi et al., 2005) , prostate cancer (Fukuchi et al., 2004; Homma et al., 2004; Kulig, Cwiklik, Jurkiewicz, Rog, & Vattulainen, 2016) , and breast cancer (Cruz et al., 2010; Gruenke et al., 1987; Lappano et al., 2011; Nelson, Chang, & McDonnell, 2014; Wu et al., 2013) . The potential relationship between COPs and several chronic diseases is summarized in Table 1 - 2 , accompanied by a brief description of the speculated mechanism(s). For more information about other chronic diseases and details regarding 7 each specific COPs, go to APPENDIX A - Table 2 . Due to the demonstrated effects of COPs in diffused and emerging human pathologies, there is an imperative demand on more accurate and systematic information about COPs intake in targeted populations ( i.e., infants, elder people, etc.). Table 1 - 2 : Summary of the table reported in Maldonado - Pereira et al., 2018 which describes the relationship between COPs and different chronic diseases . Dietary COPs are highlighted . CARDIOVASCULAR DISEASES ATHEROSCLEROSIS Oxysterol Affected tissue Effect Reference 27 - OH Peripheral artery - Oxidative stress (mixture) - Inflammation (alone) - Endothelial dysfunction/cell phenotype changes - Acts as agonists of liver X receptors (LXRs) Virginio et al., 2015 Carotid Leonarduzzi et al., 2007 Aorta Upston et al., 2002 Carotid/Aorta Garcia - Cruset et al., 2001 Coronary Vaya et el., 2001 24S - OH ND - Apoptosis (alone) - Acts as agonists of liver X receptors (LXRs) Alkazemi et al., 2008, Gargiulo et al., 2015 22 - OH ND - Vascular calcification (alone/mixture) - Acts as agonists of liver X receptors (LXRs) Alkazemi et al., 2008, Gargiulo et al., 2015 25 - OH Peripheral artery - Oxidative stress - Inflammation (alone /mixture ) - Apoptosis (alone) - Endothelial dysfunction/cell phenotype changes - Vascular calcification - Acts as agonists of liver X receptors (LXRs) Alkazemi et al., 2008, Virginio et al., 2015 - OH Carotid - Oxidative stress - Inflammation (alone) - Apoptosis (alone) - Endothelial dysfunction/cell phenotype changes (alone) Helmschrodt et al., 2013 , Leon arduzzi et al., 2007 Micheletta et al., 2004, Iuliano et al., 2003, Carpenter et al., 2003, Vaya et al., 2001 Carotid/Aorta Garcia - Cruset et al., 2001 Coronary Vaya et al., 2001 - OH Carotid - Oxidative stress - Inflammation - Apoptosis - Endothelial dysfunction/cell phenotype changes Helmschrodt et al., 2013 Carotid/Aorta Garcia - Cruset et al., 2001 , Alkazemi et al., 2008 Coronary Vaya et al., 2001 8 Table 1 - 2 CARDIOVASCULAR DISEASES ATHEROSCLEROSIS 7 - Keto Carotid - Oxidative stress - Inflammation - Apoptosis - Endothelial dysfunction/cell phenotype changes - Acts as agonists of liver X receptors (LXRs) Helmschrodt et al., 2013, Leonarduzzi et al., 2007, Micheletta et al., 2004, Iuliano et al., 2003, Vaya et al., 2001, Upston et al., 2002 Carotid/Aorta Garcia - Cruset et al., 2001 Coronary Vaya et al., 2001 - Epoxy Carotid - Oxidative stress - Inflammation (alone) - Apoptosis (alone) - Endothelial dysfunction/cell phenotype changes (alone) Helmschrodt et al., 2013, Vaya et al., 2001 Carotid/Aorta Garcia - Cruset et al., 2001 Coronary Vaya et al., 2001 - Epoxy Carotid - Oxidative stress (alone/mixture) - Inflammation (alone/mixture) - Apoptosis (alone/mixture) Helmschrodt et al., 2013 Carotid/Aorta Vaya et al., 2001 Coronary Garcia - Cruset et al., 2001, Vaya et al., 2001 Triol Carotid - Oxidative stress - Inflammation - Apoptosis - Endothelial dysfunction/cell phenotype changes (alone) - Vascular calcification Helmschrodt et al., 2013 NEUROLOGICAL DISEASES MULTIPLE SCLEROSIS Oxysterol Affected tissue Effect Reference 25 - OH Central nervous system (neurons) Damage to the myelin in the central nervous system Mukhopadhyay et al., 2017 - OH 7 - Keto CANCER BREAST CANCER Oxysterol Affected tissue Effect Reference 27 - OH Breast Promotes the proliferation of the estrogen receptor (ER) positive breast cancer cell lines in vitro. Cruz et al., 2010 Wu et al., 2013; Nelson et al., 2014 25 - OH - Induce the recruitment of Era to the ERE site located in the pS2 promoter sequence in MCF7 cells. - Induce proliferative effects in a dose dependent manner. Lappano et al., 2011 Forty five percent of Americans have suffered from some type of chronic illness ( 2014), and according to the Agency for Healthcare Research and Quality (AHRQ), chronic 9 diseases (including those COPs related diseases), are the most prevalent healthcare issues in the United States (NIH, 2017; Quality, 2020) . These conditions include arthritis, asthma, cancer, cardiovascular (heart) disease, depression, and diabetes, though these are only a few o f many chronic illnesses that negatively affect the lives of Americans ( Quality, 2020 ). The World Health Organization's Department of Evidence and Research shows that many deaths have been caused by chronic diseases some of them closely related with COPs. From a physiological point of view, individual COPs play an important role in the development of several age - related diseases because of a decrease in oxidative defenses and control of the level of oxysterols, as it is observed in hypercholesterolemia (Micheletta et al., 2004; Zarrouk et al., 2014) . Hypercholesterolemia not only can result in coronary heart disease, stroke, and peripheral arterial disease, type 2 diabetes, and hypertension atherosclerosis but high amounts of cholesterol in the body increase the amounts of COPs in the body a s well (Carpenter et al., 2003; Garcia - Cruset, Carpenter, Guardiola, Stein, & Mitchinson, 2001; Gargiulo et al., 2015; Menéndez - Carreño et al., 2011; Murakami et al., 2001; NIH, 2017; Shim et al., 2008; Upston et al., 2002; Vaya et al., 2001) . Given the different biochemical and biophysical properties of COPs in altering specific cell compartments and enzyme/receptors, several cells and tissue - specific pathological effects have been performe d. Different studies have shown that some COPs advers ely affect the function of some major organs like brain, eyes, heart, and vessels (B retillon et al., 2007; Gargiulo et al., 2016) . Understanding how the structure and chemistry of COPs define their speci fic function and mechanism(s) of action inside the human body could help to identify how COPs affect targeted tissues and the exact conditions that triggers their formation. Many hypotheses have arisen to explain the specific role that COPs play in the pat hology of a variety of diseases ( Table 1 - 2 ). 10 1.2.3 COPs as biomarkers of chronic diseases E xperimental evidence point s out that COPs can adversely affect the function of some major organs, but the interaction and mechanisms of COPs in the specific affected organ is still not well understood. (C. Y. Lee et al., 2009) have suggested that the apparent association of specific COPs with people more prone to suffer of these chronic diseases could be beneficial, as COPs could be used as biomarkers for these diseases (Aldini, Yeum, Niki, & Russell, 2010; Alkazemi, Egeland, Vaya, Meltzer, & Kubow, 2008; Iuliano et al., 2003) . As an example, Linseisen's work (Linseisen, Wolfram, & Miller, 2002) associated the - OH with lung cancer risk revealing high risk estimates, which was lately confirmed by Kang and co - workers (Kang et al., 2005) . Abnormal cholesterol biosynthesis h - OH in Smith - Lemli - Optiz syndrome (L. Xu et al., 2011; Libin Xu et al., 2012) . (Yoshida et al., 2003) found some sort of connection between oxysterols and the hepatic bile disease, proposing that the most common oxysterols in gallstones may be generated in the gallbladder in response to bacterial infection. Leoni and Caccia (Leoni & Caccia, 2011) mentioned the use of 24S - OH as a possible surrogate biomarker by the number of metabolically active neurons located in the grey matter of t he brain due to its reduced levels of 24 - hydroxylase (CYP46A1) with subsequent reduction in the formation of 24S - OH and lower efflux from the brain to the circulation (Leoni et al., 2013; Leoni et al., 2008) . These levels of 24S - OH in the circulation were found to be significantly reduced compared to controls in different neurodegenerative diseases such as Alzheimer's disease (AD), Multiple sclerosis (MS) and Huntington's disease (HD) (Leoni & Caccia, 2011; Lütjohann et al., 2000) . They also associated higher levels of 27 - OH with the processing Impairment (MCI) patients. In individuals suffering multiple sclerosis (MS), elevated 11 concentrations of 7 - keto have been detected in the cerebrospinal fluid; however, mechanistic evidence ha s been reported only for the induction of neuronal damage via the activation and migration of microglial cells (Anne Vejux & Gérard Lizard, 2009) . Other COPs derived from cholesterol auto - - - OH, and 25 - OH) also present in the cent ral nervous system are known to cause damage to the myelin; however, a complete understanding of the associated molecular mechanisms is still missing (Mukhopadhyay et al., 2017) . Huntington's disease (HD), a disease that causes neuronal dysfunction and death, has been associated to transcriptional repression, oxidative injury and mitochondrial dysfunction provoked by accumulation of enzymatic 24S - OH and 27 - OH in brain. Analyses performed postmortem on brain tissue of HD patients showed a 60% decrease in 24S - OH, 30% increase in cholesterol, and 50 70% increase in 7 - - OH (both derived predominantly from ROS action on cellular cholesterol), suggesting a significant inhibition of cholesterol m etabolism and the contribution of oxidative stress in HD pathology (Kreilaus, Spiro, McLean, Garner, & Jenner, 2016) . Lastly, for Parkinson's disease, (Bosco et al., 2006) stated that enzymatic oxysterols (mainly 24S - OH and 27 - OH, and - synuclein aggregation and destruction of dopamine - containing neurons. All these relationships c ould help to use COPs as biomarkers in different human diseases. Nevertheless, the concurrence of biochemical action ( i.e., cytochrome P450s metabolism) and oxidative stress ( i.e., cholesterol autooxidation) in the formation of individual COPs make challen ging a severe and robust identification of disease - specific markers. Thus, although a potential use of COPs as biomarker exists, this cannot prescind from an overall assessment of individual physiology and oxidative conditions. 12 1.2.4 COPs as a P romoter of C ellular A poptosis Even though there is a knowledge gained over the years about the mechanisms by which COPs exert their pathologic effects and considering that cholesterol oxidation generates more than 70 derivatives, volatile organics and H 2 O 2 (L. L. Smith, 1996) , we still lack an exhaustive comprehensio n of the mechanisms of formation and action. It is worth to mention that there is a significant overlap between COPs generated via enzymatic pathways (mainly P450's associated metabolism of cholesterol and other COPs precursors) and COPs derived from auto - oxidation of cholesterol ( Table 1 - 1 ). There is a copious amount of literature demonstrating COPs' cytotoxicity on several cell lines and in vivo. The cytotoxic activity of COPs is mainly derived from their ability to induce apoptosis through several mech anisms. An exhaustive discussion of biological action of COPs is beyond the scope of this chapter , and the author suggest s a few compelling reviews already published. Here, we will summarize the significant findings regarding the role played by those COPs which are derived from diet. Apoptosis is a programmed cell death, a critical biological process involved in ontogenesis and tissue homeostasis. Dysregulation of apoptosis can promote important diseases, including cancer and atherosclerosis (Lopez, 2015; Tr acie Seimon and Ira, 2009) . In general terms, cell apoptosis occurs via two pathways: the mitochondrial or intrinsic pathway and the death receptor - dependent or extrinsic pathway (G. Leonarduzzi, Poli, Sottero, & Biasi, 2007; Sinéad Lordan and John, 2009) . The first leads to direct activation of caspases, a family of cytosolic proteases that transmit the apopto tic pathway by making specific protein cleavages (Wolf & Green, 1999) . On the other hand, the mitochondrial pathway involves alterations of the mitochondrial potential, which triggers the production of ROS and/or mitochondrial membrane permeabilization (Biasi et al., 2009; G. Leonarduzzi et 13 al., 2007) . Mostly endogenous COPs mediate apoptosis via the mitochondrial pathway, although a few recent studies have found evidence of actions on the caspase cascade as well (Sinéad Lordan and John, 2009) . The death receptor pathway is activated through the binding of cytokine ligands to receptors of the tumor necrosis factor (TNF) superfamily, such as Fas, lymphotoxin, TNF receptor (TNFR) or TNF - related apoptosis - inducing ligand (Sinéad Lordan and John, 2009) . - OH and 25 - OH up - regulated Fas expression and induced apoptosis in vascular smooth cells when treated with oxidized LDL (oxLDL) (T. S. Lee & Chau, 2001) . - OH and 7 - keto failed to induce the proapoptotic ligand TNF - endothelial cells (HUVECs), but triggered apoptosis by activating the interleukin IL - secretion only after 24 h of incubation, suggesting a time - dependent increase after exposure (S. Lemaire et al., 1998) - OH, 7 - - epoxide also induced the release of the inflammatory cytokine IL - 8 in U937 (human promonocytic leukemia cells), monocytes/macrophages, as much as THP - 1 cells (Lemaire - Ewing et al., 2005; Y. Liu, Mattsson Hultén, & Wiklund, 1997) . Several protein kinases involved in the upstream induction and downstream execution stages of apoptosis have shown modified activity after - OH and 7 - keto (Adamczyk, Scherrer, Kupferberg, Malviya, & Mersel, 1998; Berthier et al., 2005) . The activity of protein kinase C a key enzyme of the cell activation pathway (Moog et al., 1991) - - OH in neuronal cells (Sinéad Lorda n and John, 2009) , but not in macrophages (Luu, 1991; Moog et al., 1991) , indicating a cell dependency in protein kinases' regulations. More interesting is the potential dysregulation o f mitochondrial control exerted by - OH, 7 - keto, and - epoxide ha ve shown to induce loss of mitochondrial transmembrane potential 14 (Lemaire - Ewing et al., 2005) , which is accompanied by release of cytochrome c in the cytosol (Gérard Lizard and Carole Miguet and Ginette Besséde and Serge Monier and Serge Gueldry and Dominique Neel and Philippe, 2000) . Cytochrome c , an essential hemoprotein of the respiratory chain, has an intermediate role in apoptosis by triggering caspase 9, which in turn activates caspase 7 and 3 (Wolf and Green, 1999). The cytochrome c apoptotic pathway has been observed in several cell lines, including U937 (Gérard Lizard and Carole Miguet and Ginette Besséde and Serge Mo nier and Serge Gueldry and Dominique Neel and Philippe, 2000) , MCF - 7 and MCF - 7/c3 cells (Prunet, Lemaire Ewing, M é n é trier, N é el, & Lizard, 2005) , upon exposure of 7 - - OH. Regarding caspase activation, COPs activate both initiator and effector caspases, thus exerting activity at both up and downstream steps of the apoptotic proces s. Several reports have put 7 - - OH and 25 - OH in the frontline for activation of caspase 8 and 12 (initiator caspases), as well as caspase 3 (effector caspase) (T. S. Lee & Chau, 2001) . Interestingly, several studies agree - OH has no effect on the caspase cascade (S. Lemaire et al., 1998; Lemaire - Ewing et al., 2005; Lordan, Mackrill, & O'Brien, 2009; Wolf & Green, 1999) . Other mecha nisms of cytotoxicity have been reported, including altered transport of small molecules via alteration of plasma membrane fluidity and permeabilization, cell detachment, leakage of cell enzymes, as well as interference with DNA synthesis ( APPENDIX A Tab le 2 (Jusakul, Yongvanit, Loilome, Namwat, & Kuver, 2011; Sevanian & Peterson, 1984, 1986) . The 5,6 - epoxy isomers and the derived triol have demonstrated inhibition activity towards DNA synthesis in hamster V79 cells at micromolar con centrations (Sevanian & Peterson, 1984) ; similar results have been reported for the 7 - keto, whose effect are synergic with the e poxides (Sevanian & Peterson, 1986) . It is believed that the mutagenetic effect r elates to 15 the ability of epoxides of generating ROS (Jusakul et al., 2011) . Other potential pro - carcinogenic effects, including DNA fragmentation, have been observed in Caco - 2 cells colon - - - epoxy (Biasi et al., 2013; Roussi et al., 2005) . In prostate cancer, 27 - OH can induce DNA damage, regulate cyclooxy genase - 2 expression, and stimulation of tumor cell migration (Cruz et al., 2010; Zarrouk et al., 2014) , which is accompanied by proliferation, stimulation and increase of androgen receptor (AR) transcriptional activity (Raza et al., 2016) . 1.2.5 COPs and C ell S urvival M echanisms Concurrently with pro - apoptotic triggeri ng, COPs have been lately associated with survival anti - apoptotic mechanisms (Beyza Vurusaner and Paola Gamba and Gabriella Testa and Simona Gargiulo and Gabriella Leonarduzzi and Giuseppe Poli and Huveyda, 2016) ; (Beyza Vurusaner and Gabriella Leonarduzzi and Paola Gamba and Giuseppe Poli and Huveyda, 2016) . A study by Berthier and co - workers (Berthier et al., 2005) proved that 7 - keto can delay the apoptotic effect induced by 7 - keto itself at relatively high concentrations (100 - keto seemed to transiently induce the MAPK - Erk kinase - 1 and 2 survival pathways - 1 human monocytes death by phosphorylating the Bcl - 2 antagonist of cell death (BAD), which then delays mitochondrial damage (G. Leonarduzzi et al., 2010) . A concentration - dependent behavior in U937 macrophages was also found for the side - chain enzymatic 27 - OH, derived from CYP27A1 activity (A. Vejux & G. Lizard, 20 09) /mL), 27 - OH inactivates the PI3K/Akt survival cascade via phosphorylation of Thr308 residue in Akt, whereas at higher - independent apoptosis (Valérie Riendeau and Christophe) . An analogue concentration - dependent survival signaling was 16 observed for - OH in HUVEC cells by activation of the MEK/ERK cascade (Trevisi et al., 2010) . 1.3 DIETARY COPS: O CCUR RENCE I N F OOD Historically, the evaluation of food quality and safety during processing, preservation, and storage has relayed in targeted single response studies, evaluating quality aspects after a particular treatment ( i.e., increase stability, improve texture, flavor, and digestibility), or mainly been focused on microbial reduction. On the other hand, lipid and cholesterol degradations are complex phenomena that can be addressed only by a multi - response analysis, which is h ard to achieve considering the intrinsic difficulties of monitoring several molecules possessing different chemical nature. Although several reports have been published in the last decades (Baggio & Bragagnolo, 2006; Brzeska, Szymczyk, & Szterk, 2016; D. Derewiaka & Obiedzinski, 2010; I. Medina Meza & Barnaba, 2013; Vesa M Olkko nen et al., 2017; Sarantinos, 1993; Savage et al., 2002; Sieber, 2005; Zardetto, Barbanti, & Rosa, 2014) , t he information regarding COPs content in foods is still incomplete. The limited data currently available has been obtained from studies performed in selected type of foods such as meat 2015; Rey, López - Bote, & Buckley, 2004) ; (Andrea Serra an d Giuseppe Conte and Alice Cappucci and Laura Casarosa and Marcello) , eggs (Boselli, Velazco, Caboni, & Lercker, 2001; Mazalli & Bragagnolo, 2009; Tsai & Hudson, 1984) Tsai and Hudson, 20 06), milk (Z. Liu, Rochfort, & Cocks, 2016; Rose - Sallin, 1995; Sieber, 2005) , infant formula (Przygonski, Jelen, & Wasowicz, 2000; Rom eu - Nadal, Chavez - Servin, Castellote, Rivero, & Lopez - Sabater, 2007) and few others. It is imperative to provide a database of COPs concentrations for the 17 major food products consumed by US population, with data obtained from real cooking procedures and by different population g roups. 1.3.1 Food P rocessing T riggers COPs F ormation in F oods Traditional food processing methods, such as drying, frying, steaming, and canning, involve heat treatment of the food matrix. It is well known for several macronutrients, including amino acids, carbohydrates as well as lipids, that heat conveys sufficient energy to trigger autoxidation, causing the formation of several degr adation compounds. For example, acrylamide and malondialdehyde are end - products of Maillard reaction and fatty acids lipid peroxidation, respectively. For their toxicity towards humans and their occurrence in foods, both have been classified as hazardous s ubstances, and their risk exposure in humans has been constantly monitored in both United States and Europe (Dorne, Bordajandi, Amzal, Ferrari, & Verger, 2009) . Similarly, cholesterol oxidation gives rise to several degradation products whose toxicity has gathered the attention of the scientific community worldwide. Given the susceptibility of the C5 - C6 double bond to a radical attack, heat triggers the cholesterol oxidation at temperatures as low as 100 °C (I. G. Medina Meza, Rodriguez - Estrada, Lercker, Soto - Rodríguez, & Garcia, 2011; J. - S. Min et al., 2015) . These temperatures are easily achievable with common processing and food preparation techniques. Industrial processing (i.e., cooking, pasteurizing, canning, etc.) occurs at variable times and temperatures depending on the type of process and food. Effecti ve canning of meats for example, takes place at 121 °C. Industrial frying takes place between 163 and 188 °C, depending on the type of meat; whereas in spray drying, a technique used to produce powders from a liquid, often used in the manufacturing of baby formulas, temperatures between 200 and 400 °C are used (Meister, Aebischer, Vikas, Eyer, & De 18 Pasquale, 2000; Ve rardo, Riciputi, Messia, Marconi, & Caboni, 2016) . As a matter of facts, it has been found that several cooking methods considerably increase the fo rmation of COPs in meats (Broncano, Petron, Parra, & Timon, 2009; Mar Roldan and Teresa Antequera and Monica Armenteros and Jorge, 2014) . Microwave heating (Herzallah, 20 05) , pan roasting, oven grilling (Khan et al., 2015) , oil - frying (D. Derewiaka & Obiedzinski, 2010) and other forms of cooking (Nielsen, 1996b) all affect the production of COPs. According to the US Department of Health and Human Services, meat and poultry should be roasted at a minimum of 1 62 ° C (FoodSafety.gov, 2020b) . Even proper roasting temperatures are more than sufficient to produce COPs (Shozen, 1995) . Additionally, a variety of non - traditional methods of processing, including infrared heating, microwave heating, ohmic heating, high pressure processing, ionizing radiation, pulsed electric field and ultrasound are believed to be less harsh on meat products (Avsaroglu, Buzrul, Alpas, Akcelik, & Bozoglu, 2006) ; (Ilce Gabriela Medina - Meza and Carlo Barnaba and Gustavo, 2014; Medina Meza et al., 2014) . Compared to traditional technologies, these novel thermal and nonthermal technologies reduce/elimi nate temperature exposure or decrease treatment time, but do not eliminate oxidation triggering factors all together (Herzallah, 2005; I. Medina - Meza & C. Barnaba, 2013; I. G. Medina - Meza, Barnaba, & Barbosa - Cánova s, 2014; Medina Meza et al., 2014) . However, we are far from a conclusive evidence that those novel technologies are reliable alternatives to lipid and cholesterol degradation (Ilce Gabriela Medina - Meza and Carlo Barnaba and Gustavo, 2014; Medina Meza et al., 2014) . Prepared meals like ready - to - eat foods also pose a significant r isk (Ubhayasekera, Kochhar, & Dutta, 2006) . These meals usually undergo several processing stages, from heating to pasteurization temperatures, followed by freezing, as part of the storage process before distribution; once stored for days, ready - to - eat meals finally make it home where they are re - h eated or microwaved. It has 19 been found that refrigeration as well as re - heating increase the formation of COPs (J. S. Min et al., 2016) . 1.3.2 Baby F oods Exposure to food - derived toxic compounds can be critical in sensi tive populations, especially infants and children. Powdered milk is a known source of COPs, although the reported amounts differ according to the milk process (Dionisi et al., 1998; Gabriella Leonarduzzi et al., 2005; Pickett - Bernard, 2006; Przygonski et al., 2000; Romeu - Nadal et al., 2007; Scopesi et al., 2002) . Powdered milk is manufactured using the wet blending - spray drying process in cow milk. This process begins with milk pasteurization to decrease the pathogenic bacteria. The milk is then evaporated at 77 °C and then sent to the spray drying unit. This unit pre - heats the product up to 93 °C; the product is then pumped in the spray d ryer at 138 204°C. This process provides the proper conditions for triggering hydroperoxide formation leading lipid and protein oxidation, among other reactions (Damjanovic - Desic, S., & Birlouez - Aragon 2011; Sieber, 2005) . McCluskey and co - workers found up to 60 ppm COPs in skim powdered milk (McCluskey, 1993) , whereas Scopesi et al. (2002) demonstrated that the content in 7 - keto is in baby formula is 5 - fold higher than human breast milk. Infant formula plays an important role in infant grow th and health; according to a report from the US Institute of Medicine, it may be the only source of nutrition for many infants during the first 4 6 months of life (2006). Around 2.7 million of infants by the age of three months rely on it for some portion of their nutrition (Martin, Ling, & Blackburn, 2016) . Infant formulations are enriched with several additives, including essential polyunsaturated fatty acids (PUFA), phytosterols, vitamins, which may play a synergistic role in the oxidation promotion of COPs. PUFAs are highly susceptible to 20 oxidation, and hence favored the choleste rol oxidation as well (Barnaba, Rodriguez - Estrada, Lercker, Sergio Garcia, & Medina - Meza, 2016; Barriuso, Ansorena, Poyato, & Astiasarán, 2015; Barriuso, Mariutti, Ansorena, Astiasará n, & Bragagnolo, 2016) . Among them, docosahexaenoic acid (DHA), and arachidonic acid (ARA) are the most common fatty acid of the PUFA family used in enriched milks, they are critical for the brain and retina developme nt, and therefore have an influence upon visual acuity and learning abilities (Romeu - Nadal et al., 2007) . As infants progress from formula to solid foods ( i.e., meat, eggs, cheese, etc.), their exposure to COPs will potentially increase and affect their metabolism (Sander, Addis, Park, & Smith, 1989) . Zunin et al. (2006) found considerable amounts of 7 - keto and triol in meat - based homogenates (2.3 g and 0.7 g per serving respectively), which was dram atically increased when vegetable oil was added to the formulations. 1.3.3 Food C hain: P ackaging and S torage Food products are packaged, shipped, and stored as part of their processing before retail . Packaging and storage methods can also affect the formation of oxidative products. It has also been shown that storage time can increase cholesterol oxidation (Du & Ahn, 2000; Mazalli & Bragagnolo, 2007; Nielsen, 1996a; Petrón, 2003; Pie, 1991; Tarvainen, Nuora, Quirin, Kallio, & Yang, 2015; Vore, 1988) . The content of COPs has been reported to increase by six times after 2 weeks of storage at 4°C, being higher in cooked meat than in raw food. Therefore, vacuum storage of cooked meat products has been suggested as an alternative to reduce their formation (J. S. Min et al., 2016) . Reduction in surface area of food exposed to the atmosphe re and the amount of light absorbed by the food, as well as the addition of a surface spray and light reflecting or absorbing packaging can all help reduce the formation of COPs (Khan et al., 2015; Li, Cherian, Ahn, Hardin, & Sim, 199 6; 21 Mariutti & Bragagnolo, 2017; I. G. Medina - Meza et al., 2014; Medina Meza et al., 2014; J. - S. Min et al., 2015; Overholt et al., 2016; Savage et al., 2002) . A decrease in time from farm to table could also considerably decrease their accumulation. Changes in food consumption habits can represent a challenge in terms of establishing exposure to toxic compounds derived from diet. In the United States, the consumption of ready food products have increased in the last decade. Sandwiches, hot dogs, hamburgers, wrap thawed and microwaved before consumption. Heat produced d uring microwaving of food have shown to accelerate the chemical oxidation (A ziz, Mahrous, & Youssef, 2002; Picouet, Fernandez, Serra, Sunol, & Arnau, 2007; Savage et al., 2002; Yarmand & Homayouni, 2009) , compared to traditional cooking. Home meal preparation may affect not only cholesterol and lipids, but also protein fraction and its derivatives enhancing the probability of the food matrix to undergo several oxidative transformations. Lo gistics between farmers, processors, shipping companies and retail locations could be an important step in reducing the storage time of animal food products. However, although there is some knowledge on the relationship between packaging and storage condit ions, there is still a need of experimental data regarding the effect of storage time and other storage conditions (as well as other food cooking methods and conditions) on COPs content. As previously explained, these thermal changes vary among the food m atrix, and the cooking methods and conditions; hence, COPs formation becomes not only directly related to the food matrix composition, but also in the processing method, packing and storage conditions. 22 1.4 COPS: A FOOD TOXICOLOGICAL TARGET? 1.4.1 Fate of the COPs: R isk and E xposure A ssessment Humans are exposed to a variety of substances from multiples exposure routes ( i.e., inhalation, contact, ingestion) and sources ( i.e., air, food, soil, water) (Dorne et al., 2009) . Risk is the probabili ty of an adverse effect on man occurring because of a given exposure to a chemical or mixture, while hazard is the inherent capacity of a chemical or mixture to cause adverse effects in man under conditions of exposure (Hanlon, Brorby, & Krishan, 2016) . Food toxicology has emerged as a critical topic for both the sc ientific community and governments, leading to the formation of different projects, like the Total Diet Study (TDS) led by the Food and Drug Administration (FDA, 2014) in the United States. The project started on 1961 and aims to systematically monitoring several contaminants and nutrients present in the average U.S. d iet. Unfortunately, even though special attention has been given to the analysis of the American diet, there is no specific study on the risk exposure of COPs. Currently, there are no federal regulations for food processing and storage conditions consideri ng the content of COPs and the consequent human risk exposure, even considering the broad body of evidence demonstrating the direct relationship between COPs and several chronical diseases (Del Rio et al., 2013; Mukhopadhyay et al., 2017) . T his information is essential to pursue more detailed studies on the relationship between COPs' intake and toxicological effects. The TDS is monitoring about 800 compounds in the US diet during 2014 2017 (FDA, 2014). Selected pesticides, herbicides, radion uclides, nutrients, and toxic elements are tracked year by year through 4 market basket of food products. The food database used in the TDS can potentially be a good starting point to perform a systematic monitoring of the occurrence of COPs and to estimate the annual dietar y intakes in the U.S. population. Gaining a deep understanding of how and from what sources humans consume 23 cholesterol oxidative products could provide information to help reduce consumption. A e effects of COPs related chronic diseases. From studies published decades ago, the toxicity and the potential hazard activity of single compound, performing an assessmen t of COPs exposure is challenging. Ideally, occurrence data with concentrations and frequency should be available in exhaustive, consistent lists. In practice, these conditions rarely met. The most common information on food consumption is derived from die tary surveys, usually conducted on a representative sample of individuals. Some surveys are based on food - frequency, dietary - history questionnaires, or household purchases without cover longer periods of times in term of individual dietary consumption (Dorne et al., 2009) . Geographical and cultural differences can influence exposure levels of COPs. Because of their diet rich in fats and meat products, the U.S. population may be exposed to high cholesterol diet, and therefore its oxidative products, at a very early age. In addition, indi viduals, and particular subpopulations (i.e., infants, children, and elder people) may be exposed or respond differently to these chemicals (Cote et al., 2016) . Data sets regarding exposure and effects are needed to evaluate the dose - response (toxicological assessment) and further risk characterization of COPs. For a priori hazard assessment a dietary exposure model should be performed. A combination of e xposure (total dietary intake) and kinetic modelling (dose - depending activity) could give the initial hints in the estimation of human exposure to COPs. Informing industry members, stakeholders, and federal agencies of the dietary intake of COPs and their negative health effects is crucial to take the proper actions in reduce its production during the whole food chain. 24 1.4.2 COPs as B iomarkers of F ood S afety Several omics ( i.e., metabolomics, lipidomics, and nutrigenomics, among others) have been already applie d to characterize postharvest metabolite heterogeneity of fruits (Romina Pedreschi and Pablo Muñoz and Pau la Robledo and Cecilia Becerra and Bruno, 2014) , food toxins , and nutritional components (Vergères, 2013) for studying biomarkers (Bordoni & Capozzi, 2015) , as we ll as metabolic pathways (Jain, Dürr, & Ayyalusamy, 2015) . In the food area, foodomics and metabolomics profiling have been mainly focused on food quality, safety, and nutrition (Sooah Kim and Jungyeon Kim and Eun Ju Yun and Kyoung Heon, 2016) ; nonetheless the application of omics to evaluate and trace effects of processing as fingerprint of the process op erations themselves is scarcely used. The assessment of dietary level of cholesterol and its derivatives combines dietary consumption data with occurrence. Cholesterol information should be found in the nutritional label that the FDA requests for the food products derivatives. This information may be insufficient for a consistent and systematic monitoring of COPs content in food, to support a risk assessment study. Challenges in cholesterol and COPs quantitation has been reduced by applying omics approa ches to the field, i.e., cholesterolomics, a field that is dedicated to the extraction, isolation, and quantification of the cholesterome in food, cells, tissues, organs and biofluids ( (Griffiths et al., 2017) . Chromatographic techniques couple with single (GC - MS) or triple quadrupole (LC - MS/MS) have been enhancing COPs detection and quantification (Chiu, 201 8; Helmschrodt et al., 2013) . 7 - Keto has been largely detected and quantified in both model and food systems (Rodriguez - Estrada, Garcia - Llatas, & Lagarda, 2014) . During thermal - induced oxidation, hydroperoxides in C - 7 are mainly generated, thus 7 - keto is one of the most representative COPs in food systems ranging from 25 30% to 70% of the total content of COPs. This has suggested that 7 - keto can be the most reliable biomarker of cholesterol oxidation due high temperature processing of foods (Rodriguez - Estrada et al., 2014) . However, other processing such as ultra - high temperature (UHT) pasteurization can also generate sidechain COPs (Pikul et al., 2013) , including the demonstrated cytotoxic 25 - OH. It is still unclear if COPs composition can reflect specific processing technologies, as some early studies have demonstrated. For example, gamma radiation triggers the specific ox idation of cholesterol in the positions C5 and C6 (Maerker & Jones, 1993a; Ilce Gabriela Medina - Meza et al., 2012) . However, more mechanistic, and quantitative studies for a suitable estimation of COPs in food products, and a harmonization of methodologies for their determination is still needed. 1.5 PARADIGM SHIFTS AND FUTURE CHALLENGES 1.5.1 Oxidative M echanisms: H olistic A pproach Many conditions are responsible of trigging/ inhibiting the formation of cholesterol oxidative products in food preparations, as reviewed in APPENDIX A, Tables 3 7. Given the discussed complexity on the oxidative phenomena in the food matrix, any predictive model that would aim to considers all chemi cal and biochemical parameters, as well as intrinsic the oxidative process considering a few factors at the time. Biomimetics and model systems have been widely used to reproduce several aspects of the cell or tissues outside a living system and are useful to represent a real - world phenomenon. These experimental settings may help to understand the biochemistry associated to the formation of COPs. Models for effec ts of heating on cholesterol oxidative product formation (Chien et al., 1998) and for high pressure processing's effects on cholesterol oxidative pr oduct formation (Ilce Gabriela 26 Medina - Meza & Barbosa - Cánovas, 2015; Medina Meza et al., 2014) have been already developed. Further model systems that can account for raw material composition and heterogeneity, processing and storage time and conditions, presence/absence of antioxidants: together, this information could also be very beneficial in a ssessing COPs risk exposure. On the other hand, cell biomimetics and animal studies can effectively provide information regarding COPs physiological absorption. Model systems not only can help to predict the concentrations of cholesterol oxidative product s in certain foods, tissues, cell lines and body fluids but also can be used to further understand human exposure to cholesterol oxides (Egeghy et al., 2016) . In our opinion, a holistic approach in the cholesterol and lipid oxid ation fields should include targeted and high throughput analytical techniques along with higher (Tara Grauwet and Liesbeth Vervoort and Ines Colle and Ann and Marc, 2014) for fingerprinting of food/biological markers and exposure assessment will potentially address the following questions: What is the association between dietary COPs and the major chronic diseases? Are COPs just foo d biomarkers? Given the power of holistic and as consequently translational approaches in the cholesterolome development, the improvement of analytical resolution and sensitivity, markers fingerprinting, and modelling is highly encouraged. 1.6 CONCLUSIONS COPs are formed both chemically and enzymatically in foods and the human body. More holistic research is required to assess the contribution of dietary and endogenous sources to the total COPs levels found in animals and humans. The need of a systematic re view of the toxicology activities of COPs and the estimation of daily intake of COPs is mandatory, to understand the connection between diet, food manufacturing and 27 a culture of prevention towards cholesterol and lipid oxidation in foods should be encouraged, together with the preservation of intrinsic natural antioxidants, as a strategy that can dampen the formation and accumulation of COPs in foods. 28 CHAPTER 2 : PHYTOSTEROLS AND THEIR OXIDATIVE PRODUCTS IN INFANT FORMULA T he content of this chapter has been previously published in th e Journal of Food Process Engineering and is an equal contribution between authors: Alice Kilvington and Lisaura Maldonado - Pereira . DOI: 10.1 111 /j fpe . 13151 2.1 INTRODUCTION Phytosterols also known as plant sterols (PS), are natural compounds which are members of the triterpene family. The triterpene group includes more than 4,000 compounds and over 100 of those are phytosterols. In food matrices, PS have been chemically characterized and quantified for over three decades (Moreau, Whitaker, & Hicks, 2002) . PS are 28 - or 29 - - hydroxyl group, and a double bond in the C5 C6 position. Compared to cholesterol , PS contain an extra methyl group, ethyl group, or double bond with a side chain of 9 10 carbon atoms in length, instead of a C8 cholesterol side chain. In plant cells, PS are primarily encountered in the plasma membrane, specifically in the outer membran e of mitochondria and the endoplasmic reticulum. They play an important role as a structural molecule, providing rigidity to the cell membrane by promoting an increase in the sterol/phospholipid ratio that is associated with membrane stiffness (Alemany, Barbera, A legría, & Laparra, 2014; Comunian & Favaro - Trindade, 2016; Moreau et al., 2002) . PS are components of all foods of vegetable origin and are known for their beneficial property in health of lowering serum total cholesterol concentration, as well as the low - density lipoprote ins (LDLs) concentration (Lagarda, García - Llatas, & Farré, 2006) . Therefore , for over the past 15 years, PS have been incorporated into several functional foods as a supplement (García - Llatas & Rodríguez - Estrada, 2011) . The structure of the most common phytosterols is shown in Figure 2 - 1. The 29 - sitosterol, campesterol, stigmasterol, and - - sitosterol is the most abundant and has a proportion of total sterols content of 60 70% (Berger, Jones, & Abumweis, 2004; Verhé, Verleyen, Hoed, & Greyt, 2005) . Figure 2 - 1 : Structures of the most common phytosterols obtained from Kilvington et al. 2019. 30 2.2 PHYTOSTEROLS FOOD SOURCES Vegetable oils are the major sources of PS in foods. Palm, sunflower, corn, coconut, rapeseed, and soybean oils have the highest content of total sterols; for example, rapeseed oil has a PS content ranging from 646 to 808 mg/100 g in fresh weight (FW); oil - based products like margarines have a content ranging from 130 to 540 mg/100 g FW (Normén, Ellegård, Brants, Dutta, & Andersson, 2007) . Other food items such as cereals (corn, rye, wheat, barley, and oats) range from 80 90 mg/100 g FW (Ryan, Galvin, O'Connor, Maguire, & O'Brien, 2007) . Additionally, cereals derivatives and nuts are great source of PS, containing the highest quantities of phytosterols compared with other foods. Another important source of PS are fruits and berries, which are popular and highly consumed due their many beneficial nutritional properties such as their antioxidant capacity, with content ranging from 6 to 75 mg/100 g FW (Piironen, Toivo, & Lampi, 2000) . PS dietary uptake varies greatly depending on the region, country and/or culture. In Northern Europe, up to 40% of dietary PS derive from cereals and cereal - based foods. However, in countries having greater availabili ty of fresh fruits the main contributor to PS in the diet are fruits and vegetables, contributing as much as 35% of dietary PS, as a study performed in Uruguay showed (Lea, Hepburn, Wolfreys, & Baldrick, 2004; Piironen et al., 2000) . Databases of almost every type of food have been developed over the years, and even country specific food items have been analyzed such as the ones reported by (Normén et al., 2007) which refers to the number of PS found in spreads, oils, seeds, and vari ous other fatty foods typically consumed in Sweden and the Netherla nds . PS content in foods has been summarized in Table 2 - 1. Animal origin food items are included because of the addition of vegetable oils or ingredients as part of different food processing techniques. 31 However, the previously mentioned variability of PS c oncentration in animal origin food items, based on the plant's origin of the additive is strongly noticeable between food items. Some differences are due to the supplementation of plant - based oils or even because of the various diets of cows, pigs, chicken s, and other animals typically consumed by humans, which contain plant products. 2.3 HEALTH BENEFITS OF PHYTOSTEROLS The chemical similarity of PS to cholesterol has played an important role in the study and analysis of the physiological aspects of PS. Most of its chemical properties have been studied based on the information known for cholesterol (Maldonado - Pereira, Schweiss, Barnaba, & Medina - Meza, 2018) . Absorption of PS in humans is low compared to cholesterol, ranging from 2 to 5% of total intake, versus 60%, respectively, (Alemany et al., 2014; Mellies et al., 1976) performed a study on infants and children to see the effects of dietary phytosterols. They found that infants fed with infant formulas enriched with - sitosterol that were three to fiv e times higher than infants fed with breast or cow's milk. At the time when this initial quantitative assessment was performed, little was known about the potential biological implications of phytosterols in infants (Mellies et al., 1976; R. Ostlund, Racette, Okeke, & Stenson, 2002) . Due to their structural similarity to cholesterol, PS were first and foremost studied for their cholesterol absorption inhibition properties. They are well known for their ability to reduce cholesterol absorption (Nestel, Cehun, Pomeroy, Abbey, & Weldon, 2001; R. Ostlund et al., 2002; R. Ostlund, Racette, & Stenson, 2003; R. E. J. Ostlund, 2002) , which is reflected in a reduced cholesterol plasma concentration (Law, 2000; Nguyen, 1999; Piironen et al., 2000; Pinedo et al., 2007) ; lipoprotein oxidation reduction anti - inflammatory 32 properties (Gab ay, Lamacchia, & Palmer, 2010) , anti - cancer properties, and apoptosis induction, positive regulation on testosterone metabolism (Awad, Hernandez, Fink, & Mendel, 1997) , cancer cell proliferation reduction by angiogenesis inhibition (Lea et al., 2004; Shahzad et al., 2017) , and tumor growth reduction (Danesi et al., 2011; Llaverias et al., 2013) . Table 2 - 1 : Summary table of the phytosterol content in oils and foodstuff published in Kilvington el at. 2019 Oils Food Processing conditions Phytosterol Concentration Reference Sunflower N/A Sitosterol 233 ± 4 mg/100 g Lin et al. (2017) Campesterol 34 ± 0 mg/100 g Stigmasterol 32 ± 0 mg/100 g Sitostanol 12 ± 0 mg/100 g Rapeseed N/A Sitosterol 378 ± 13 mg/100 g Campesterol 290 ± 10 mg/100 g Brassicasterol 83 ± 2 mg/100 g Sitostanol 59 ± 1 mg/100 g Dairy Milk N/A Total phytosterols 0.156 ± 0.00 g/100 g Srigley and Haile (2015) Eggs N/A Total phytosterols 20.7 mg sterols/100 g ingredient Menéndez - Carreño, Knol, and Janssen (2016) Meat and Poultry Meat (steak, roast beef, stew, chicken, pork, and minced meat) N/A Total phytosterols 3.47 14.9 mg sterols/100 g ingredient Menéndez - Carreño et al. (2016) Seafood Fish (salmon, shallow - fried cod, microwaved cod, and fish sticks) N/A Total phytosterols 2.01 51.9 mg sterols/100 g ingredient Menéndez - Carreño et al. (2016) 33 Table 2 - 1 Others margarine - Control - Storage (18 weeks @ 4 C) - Storage (18 weeks @ 20 C) Brassicasterol - 0.82 ± 0.18 g/100 g spread (control) - 0.71 ± 0.06 g/100 g spread (18 weeks @ 4 C) - 0.58 ± 0.05 g/100 g spread (18 weeks @ 20 C) Rudzinska, Przybylski, and (2014) Campesterol - 2.95 ± 0.23 g/100 g spread (control) - 2.12 ± 0.18 g/100 g spread (18 weeks - @ 4 C) - 2.04 ± 0.18 g/100 g spread (18 weeks - @ 20 C) Light mayonnaise N/A Total phytosterols 0.686 ± 0.01 g/100 g S rigley and Haile (2015) Spread/margarine 1 0.788 ± 0.01 g/100 g Spread/margarine - 2 0.753 ± 0.01 g/100 g Spread/margarine 3 2.88 ± 0.02 g/100 g Spread/margarine 4 3.82 ± 0.04 g/100 g Orange juice 0.423 ± 0.01 g/100 g Protein shake 1 0.529 ± 0.02 g/100 g Protein shake 2 4.86 ± 0.60 g/100 g Instant coffee 6.96 ± 0.69 g/100 g Dietary chew 1 7.73 ± 0.11 g/100 g Dietary chew 2 9.61 ± 0.09 g/100 g The National Cholesterol Education Program recommends adding 2 g/day of phytosterols to the diet to reduce LDL cholesterol concentrations and coronary heart disease risk. In 2000, the FDA issued an interim rule allowing the claim that plant stanyl and ster yl esters - containing foods reduce the risk of coronary heart disease, because their demonstrated cholesterol - lowering effect (Golley & Hendrie, 2014; Moreau et al., 2002) . Moreover, PS success in health has been proved by the development of different patents and commercial PS products currently being marketed worldwide (García - Llatas & Rodríguez - Estrada, 2011) . 34 2.4 PHYTOSTEROLS OXIDATION PS are particularly susceptible to oxidation due to their surface a ctivation property, by exposure to UV light, high by 2 5%, increasing the oxidation as consequence. The presence of heat, light, metal contaminants catalyze radical - mediated oxidation at the double bond, starting an autocatalytic oxidative chain reaction. Similarly, to general lipid oxidation processes, PS oxidation evolves through three main steps: the initiation corresponding to the generation of highly reactive radical species, the propagation of radical species via autocatalysis, and the termination of the reactions with consequent formation of thermodynamically stable compounds (Johnson & Decker, 2015) . In foods, free radicals can be generated by photosensitization mediated by either chlo rophyll (vegetable matrices; (I. G. Medina - Meza et al., 2014) or heme (animal matrices), which leads to the formation of singlet oxygen, or by reacting with metals (Boatright & Crum, 2016) . Oxidation can also occur by pre - existing reactive oxygen species (ROS) and oxidative enzymes such as cytochrome P450, superoxides, and peroxidases naturally pres ent in vegetable matrices (Ryan, McCarthy, Maguire, & O'Brien, 2009) . The oxidation of PS in food follows similar chemical pathways to cholesterol oxidation, and the products of PS oxidation are known as phytosterol oxidation products (POPs; (I. G. Medina - Meza & C. Barnaba, 2013) . PS major oxidation pathways are shown in Figure 2 - 2. Primary products of oxidation are the allylic 7 - hydroperoxides, which - - hydroxy by epimerization. Hydroxy ls can be further oxidized to the chemically stable 7 - keto compounds, - and - epoxy compounds. The epoxides generally are hydrolyzed to form the 3,5,6 - triols (Barriuso, Otaegui - Arrazola, Menéndez - Carreño, Astiasarán, & Ansorena, 2012; Cercaci, Rodriguez - Estrada, Lercker, & Decker, 2007; 35 González - Larena et al. , 2011; Lin et al., 2018; Lin et al., 2017; O'Callaghan, McCarthy, & O'Brien, 2014) . Main factors promoting POPs formation in foods are: (a) processing temperat ure and time, (b) storage temperature: affects only when temperature is over 34 C, (c) sterols structure, (d) esterification, (e) degree of saturation, and (f) food matrix (Danesi et al., 2011) . The concentration of POPs in various food products is shown in Appendix B Table 2 . POPs formation increases proportio nally to temperature and time; POPs formation is also Figure 2 - 2 : Figure published in Kilvington et al. 2019 describing the general phytosterol oxidation pathway where R denotes a specific phytosterol side chain. 36 affected by water content and oil drop size in the food matrix (Cercaci et al., 2007; McClements, Decker, & Weiss, 2007) . Moreover, w hen producing rapeseed oil industrially, it was found that the refined oil had over double the POP content (Rudzinska, M., Uchman, & Wasowicz, 2005) . 2.5 P OPS BIOLOGICAL TOXICITY Few studies have given some insight about POPs and their biological activity both in humans and animal models. Studies performed in animals have demonstrated that POPs can be absorbed at a higher rate compared to phytosterols (A. Grandgirard, Sergiel, Nour, Demaison - Meloche, & Giniès, 1999; Meynier, Andre, Lherminier, Grandgirard, & Demaiso n, 2005) . POPs distribution and accumulation in different animal tissues (aorta, heart, kidneys, liver) revealed the hig hest concentration of hydroxyl derivatives in the liver (Bang, Arakawa, Takada, Sato, & Imaizumi, 2008; Liang et al., 2011; Tomoyori et al., 2004) , but triols were also found in the liver, kidney, and the heart lipid fraction. For example, lymphatic recoveries for campesterol oxide s (16%) and sitosterol oxides (9%) were higher than for campesterol - sitosterol (2% ) ; (A. Grandgirard, Demaison - Meloche, Cordelet, & Demaison, 2004; A. Grandgirard, Martine, et al., 2004) . These trends are similar to the absorption of non - oxidized phytosterols, implying that increasing the side chain length of either the PS or POPs, decreased their absorption and that the type of oxidation relates to the degree of absorption (Ryan et al., 2007) . POPs may be related to the inflammation processes, dyslipidemia, atherosclerosis, apoptosis, and cell toxicity (Alemany et al., 2014; Y. Hu et al., 2018; P. Zunin, Calcagno, & Evangelis ti, 1998) . Adcox, Boyd, Oehrl, Allen, and Fenner (2001) demonstrated that POPs affect protein synthesis and damage the cell membrane, while measuring total protein 37 content and LDL leakage (Adcox, Boyd, Oehrl, Allen, & Fenner, 2001) . Recently, it has been hypothesized that due to the structural similarity between POPs and cholesterol oxidation products (COPs), POPs could potentially contribu te to the onset of metabolic and neurologic diseases by an irreversible accumulation in the central nervous system. A recent study - hydroxysitosterol can pass through the blood brain barrier (Schött et al., 2017; Schött et al., 2015) . Maguire, Konoplyannikov, Ford, Maguire, and O'brien - sitosterol have shown similar patterns of toxicity towards a human monocytic cell line (U937) as the cholesterol - derivative - mixture (120 adenocarcinoma (Caco - - keto and - sitosterol were moderately cytotoxic (at 60 lines; the mode of cell death was apoptosis in the U937 cell line and necrosis in the Caco - 2 and HepG2 cells (Ryan et al., 200 7) . 2.6 INFANT FORMULATION: A CRITICAL NEED food which purports to be or is represented for special dietary use solely as a food for infants by reason of its simulation o f human milk or its suitability as a complete or partial substitute for human milk (FDA, 2016) . IF provides all nutrients and sustenance for the growth and development of infants when breast feeding is not an option for physiological or medical reason s (Green Corkins & Shurley, 2016; Su et al., 2017; Vandenplas, Zakharova, & Dmitrieva, 2015) . 38 Different health issues, such as allergies and metabolic disorders, that infants could develop during their first months of life, are targeted in the production of numerous types of formulas that provide the appropriate nutrition for every child, adding or removing certain components of the infant formula based on the specific need of the infant (Maldonado, Gil, Narbona, & Molina, 1998) . In the United States common types of infant formula are cow's milk - based, soy - based, lactose - reduced , or partially hydrolyzed, and specialty. These special formulas are developed for infants who have certain conditions, like protein sensitivity, acid refl ux , pre - term, phenylketonuria, and so on. (Rossen, Simon, & Herrick, 2016) . Since the infant's nutrition plays a crucial ro le in development both short and long term (M. Lemaire, Le Huërou - Luron, & Blat, 2018) , this is the main reason why the FDA issued a Code of Federal Regulations (Title 21, Chap ter I, Subchapter B, Part 107) where the requirements are outlined for the composition of IF. As it is stated by the FDA, for each 100 - cal serving there must be 300 mg of Linoleic acid and 1.8 19). In addition, IF contains around 20% fat, which comes from vegetable oils like sunflower, palm, soy, or coconut oils (5, 8, 7, and 5%, respectively; (S. Damjanovic - Desic & Birlouez - Aragon, 2011; Hamdan, Claumarchirant, Garcia - Llatas, Alegría, & Lagarda, 2017; Hamdan, Sanchez - Siles, Garcia - Llatas, & Lagarda, 2018; Rajasekaran & Kalaivani, 2013) . PS content in vegetables oils normally added to IF can be seen in Figure 2 - 3. Sterols content in human milk differ from IF's sterols content. The animal sterols, including cholesterol, range from 12.0 to 16.6 mg/100 mL and 0.4 5.47 mg/100 mL in human milk and IF, respectively. The phytosterols content is 0.02 mg/ 100 mL in human milk versus 2.45 - sitosterol and stigmasterol (Hamdan et al., 2017) . IFs are manufactured to mimic the human milk as close 39 as possible, but many differences remain, bringing up a nutritional concern because of its effect on the baby's health. For example, fat in human milk makes up 50% of the total energy: to match that percentage, I F s need to be supplemented with external fat sources (Y. S. Chen, Aluwi, Saunders, Ganjyal, & Medina - Meza, 2019; Hageman, Danielsen, Nieuwenhuizen, Feitsma, & Dalsgaard , 2019) . Other components naturally present in human milk (hormones, vitamins, and essential fatty acids) are also supplemented in IFs to achieve the correct infant dietary Figure 2 - 3 : Table published in Kilvington et al. 2019 describing the phytosterol concentration reported in infant formula 40 requirements (Y. S. Chen et al., 2019) . An interesting case is represented by long chain fatty acids (arachidonic acid, eicosapentanoic acid, and docosahexaenoic acid), which play a crucial r ole in cognitive and retinal development. IF with a fat composition that comes only from vegetable oil has higher levels of monounsaturated fatty acids and lower levels of medium chain fatty acids compared to human milk (Hageman et al., 2019) . Recently , long chain polyunsaturated fatty acids, otherwise present in human milk, have been recently added to IFs as well (Y. S. Chen et al., 2019; Uauy & Dangour, 2009) . As it is stated above, there is significant evidence of the abundant presence of PS in IF, however, little is known about the effects of processing techn ologies on its oxidation (García - Llatas et al., 2008; Lagarda et al., 2006) . The POP content in IF can be seen in Appendix B - Table 4. Because of the high lipid content i n IF lipid due to fat supplementation with vegetable oils, oxidation is likely to occur. Few studies have investigated the formation of POPs within IF. Boatright and Crum (2016) tested three different infant formulations commonly found in the store and found that hydrogen peroxide, which is one of the ROS that can lead to lipid oxidation, was generated when preparing the f ormula according to the manufacturer's directions. Since the formation did not occur until after mixing with water, they concluded that the hydrogen peroxide was generated via a redox - cycling reaction from the initial ingredients inside the IF. This type o f study emphasizes the need of perform more research focused on the importance of IF and the effects of its manufacturing in the formation of POPs. New studies have started questioning the high PS content in IF, and hence, its potential oxidation process o ccurring during the infant formula manufacturing which generates POPs. Berger et al. (2004) recommended that children under five should not be 41 given phytosterols since they should have high cholesterol in their diets instead (Lemaire et al., 2018). Therefo re, a question that arises is: Do we need to modify the IF recipe or the manufacturing process to dampen its lipid oxidative load? 2.7 PROCESSING TECHNOLOGY RELATED TO IF Like most foods nowadays, IF undergoes rigorous processing prior to commercialization to guarantee the safety of the final product. IF is usually made by modifying cow's milk, with a variation in the whey - to - casein ratio (70/30). Whey proteins are predomin ant in breast milk (60% whey and 40% casein) and are believed to be more easily digested. Other substances that can be added are prebiotics (to aid digestion) and nucleotides. The former is normally found in breast milk and promotes brain and eye developme nt (S. Damjanovic - Desic & Birlouez - Aragon, 2011; Traves, 2019) . In general, the manufacturing process of IF involves a combination of different temperat ures, pressures, and times, with a wide range varying between 60 and 200 C, 0.8 20 MPa, and 30 s to 6 min, respectively. All these differences in the manufacturing procedures promotes the oxidation of the phytosterols present in the food matrix and produce potentially toxic compounds, such as POPs. A general IF manufacturing process is shown in Figure 2 - 4 . The main process steps are mixing, evaporation, and drying (Jiang & Guo, 2014) . Temperature and pressure are the major parameters affecting the evaporation and drying processing steps. Evaporation, a critical process step for the removal of water, is preferred to spray drying si nce it requires less energy. More importantly, milk powder produced from evaporated milk has a longer shelf life and larger powder particles with a smaller amount of included air. Jiang and Guo (2014) explain that even though milk is commonly dried by roll er drying or spray drying in a stream of hot air, spray drying is more commonly used for infant formula because roller 42 dried products have a lower solubility in water, are susceptible to irreversible component changes during drying, and because roller drie d powder has a lower microbiological quality than spray dried powder. Even though the overall manufacturing process of IF is basically the same, differences in temperatures and other processing parameters can still be observed. A patent filed in 1987 by Angel Gil and Luis Valverde, revealed the exact process used in the manufacturing of IF. The process starts mixing vegetable oil and aqueous products while heating to homogenize and emulsify the contents. Then the addition of nucleotides and other ingredie nts followed by pasteurized takes place at 95 100 C. Next step uses vacuum to condense the mixture, with a slowly dry at a low temperature, then sterilized it twice at 121 C or once at 151 C for a few seconds (Gil, A., & Valverde, 1982) . Another patent also states that infant formula is mixed and homogenized using high pressure and it is then spray dried at temperatures around 200 C (Van Den Brenk, Van Dijke, Van Der Steen, Moonen, & Van Baalen, 2015) . Moreover, Van Dijke, et al.'s patent describes the addition of a second emulsification step, pasteurization, or heat treatment of the aqueous phase from either 60 to 100 C, or 70 to 90 C, and more preferably to 85 C, with a holding time of 1 s to 6 min, more preferably 10 s to 6 min, and even more preferably from 30 s to 6 min (Van Dijke, SCHRÖDER, USTUNEL, Reinhold HALSEMA, & Moonen, 2018) . Moreover, they recommend that the lipid phase should be liquid at the temperature(s) used during the process. However, if the lipid phase is solid due to its composition it is preferably heated to above the me lting temperature of at least one lipid, preferably vegetable lipid, contained in the lipid phase, specifically to a temperature above its melting point from 55 to 60 C. 43 It is important to note that besides the variation of parameters involved in the manufacturing process previously mentioned, special attention should be given to other potential ox idation factors such as the IF packaging, handling, and preparation techniques at home that could promote the formation of POPs. 2.8 THE CHALLENGE OF FOOD PROCESSING: LIPID OXIDATION Western diet contains levels of 150 - sitosterols, campesterol, stigamasterol and traces of saturated phytostanols (Ryan et al., 2009) . Theref ore, POPs intake should be monitored to ensure low accumulation of these molecules in the human body, especially in infants, since like all unsaturated lipids, PS oxidation can Figure 2 - 4 : Infant formula milk powder manufacturing process (Kilvington et al. 2019) 44 occur during the processing, preparation, and storage of IF (O'Callaghan et al., 2014) . During a study of sterol oxidation in IF, it was found that the phytosterols were more oxidized than their animal counterpart, cholesterol (García - Llatas & Rodríguez - Estrada, 2011; González - Larena et al., 2011) pointing out the need of surveillance for these types of food. On the other hand, several studies have addressed the reduction of POPs with addition of antioxidants such as tocopherol, butylated hydroxytoluene (BHT), and plant ethanolic extracts (Rudzinska et al., 2005) ; however, no efforts have been dedicated to evaluating the different processing technolog ies and their effects in promoting PS oxidation in IF. It is imperative to build a strong scientific information base regarding not only PS consumption and its oxidized derivatives, but also their toxicity and exposure on a vulnerable population like infan ts. Lipid oxidation is a major concern, but other nutritional components can also undergo reactions during food processing. The Maillard reaction is a well - known reaction that occurs in food during thermal processing. A reducing sugar and amino acid react together to form various Maillard reaction products (MRPs), depending on the conditions. The Maillard reactions leads to decreased nutritional value of proteins and some products may have adverse health effects. Milk and milk derivative products, like infa nt formula, are high in protein and sugar and so thermal processing of milk will lead to the formation of MR Ps (Tamanna & Mahmood, 2015) . Proteins can also undergo oxidation if exposed to heat, light, or metal. These oxidation reactions can lead to racemization of amino acids. This can lead to major changes in the protein's p roperties, activity, and structure which could also cause increased toxicity (Y. S. C hen et al., 2019) . Lipid oxidation products can also react further with amino acids (Hematyar, Rustad, Sampels, & Kastrup Dalsgaard, 2019) . The processing of infant formula can have drastic effects on the composition of the 45 ingredients inside. High heat leads to lipid and protein oxidation, as well as the Maillard reaction. These reactions can lead to the formation of a wide range of components that can further react and may have drastic effects on infants. 2.9 CONCLUSION S PS are plant sterols present in vegetable food sources. Their oxidation is promoted by temperature, storage, and processing, generating oxidized derivatives know as POPs. IF is given to infants at a ver y early age, especially those who have special dietary restriction or medical conditions and cannot fulfill their nutritional needs from breast milk. These young children rely on the contents of infant formula to obtain the nutrition needed for a healthy a nd complete physical development and growth. However, the scarce information related with POPs, the mechanisms these compounds undergo, and the potential adverse health effect on infants that could result from their intake through infant formula consumptio n is still unclear and should be of great concern to the scientific community. What is the outcome when the only meal these young children have is filled with oxidized sterols that could affect them for the rest of their life? Even though there are not eno ugh studies of POPs formation due to food processing, there is no doubt that the processing of IF leads to the formation of oxidized sterol products. Therefore, high amounts of phytosterols in infant formula could result in high concentrations of POPs. The effects of infant formula processing in the POPs formation mechanism and their connection with infant's health are still widely unknown. However, it is well known that they can have consequences on human health based on studies performed in different food matrices. A deeper analysis of the processing and treatment of infant formula is crucial to obtain a better understanding of these 46 compounds' formation pathways and their biological mechanism in infants, which are a critical and vulnerable population. 47 CHAPTER 3 : EVALUATION OF THE NUTRITIONAL QUALITY OF ULTRA - PROCESSED FOODS (READY TO EAT + FAST FOOD): FATTY ACID COMPOSITION The content of this chapter has been submitted for revi sion to the Food and Chemical Toxicology Journal. DOI: 10.1101/2021.04.16.21255610 3.1 INTRODUCTION Dietary lipids provide up to 42% of the calories ingested in the Western diet, whereas nutritional recommendations of fat for adults are 20 35% (Niot, Poirier, Tran, & Besnard, 2009) . In the Western diet, ~95% of dietary lipids are comprised of triacylglycerols, ( mainly long - chain fatty acids (LCFA)), and the remaining 5% includes phospholipids (4.5%) and cholesteryl esters (0.5% the consumption of high amounts of processed meats (F. B. Hu, 2002; Schulze & Hu, 2002) . Nearly 60% of the calories consumed by the average North American individual are obtained from the intake of ultra - processed foods (UPFs) (Martínez Steele et al., 2016; Urban et al., 2016) . Fast foods (FF) (including small, large, and non - chain res taurant) and Ready to Eat . A healthy human with an average of 70 kg of body weight, stores around 141,000 kcal as fat as compared to 24,000 kcal as protein and 1,000 kcal as carbohydrate (Wang, Liu, Portincasa, & Wang, 2013) . Furthermore, dietary fat is the most calorically dense macronutrient, supplying 9 kcal/g, about d ouble of what is contributed by either protein or carbohydrate at 4 kcal/g. The increase of consumption of dietary fat from UPFs related to a qualitative imbalance (excess of saturated fatty acids and cholesterol) has been associated with the increased risk in the development 48 of several chronic diseases such as obesity, diabetes, cancer, and cardiovascular disease (Zinocker & Lindseth, 2018) . Food processing does not represent an issue for human nutrition, neither does it inherently cause negative health outcomes. However, the over - processing of food can lead to the accumulation of harmful substances formed because of thermal treatments, light exposure, storage, and aging food, the denaturalization of proteins, vitamins, and bioactive components, compromising the overall nutritional quality. Processed foods (PFs) and UPFs are food it ems considered as inexpensive, highly processed, rich in calories, but low in some essential micronutrients such as mineral and vitamins. Studies have shown that UPFs comprise 57.9% of the energy intake (Martínez Steele et al., 2016) . UPFs are mostly consumed away from home and have been proposed as a m ajor contributor to the energy intake rise (Urban et al., 2016) . Another big group of meals highly consumed within the Western diet are the RTE meals. RTE m eals are defined as any food that is either normally eaten in its raw state or has been somehow processed, for which it is reasonably foreseeable that the food will be eaten without further processing that would significantly minimize biological hazards (FDA, 2020) . Usually, RTE foods are packed processed products for sale which require minimum preparation at home. Most foods from the Western diet, including FFs and RTE meals, belong to the PFs or UPFs category according to the NOVA ( not an acronym ) classification system (C. A. Monteiro, Cannon, Lawrence, Costa Louzada, & Pereira Macha do, 2019) . These terms differ in the number of processing techniques applied during the production of the food item. NOVA is recognized by the Food and Agriculture Organization (FAO) and Pan - American Health Organization (PAHO) as a valid tool for nutrit 49 foods according to the extent and purpose of food processing, rather than in terms of (Juul et al., 2018; Martínez Steele et al., 2016; Moubarac et al., 2017) . perspective, food processing emerges as a critical topic for public health linking nutrit ional quality and food safety. The aim of this chapter is to report the fat content and fatty acids profile of a selection of UPFs, to establish a database useful not only for nutritional and clinical interventions, but also to improve food chemical safety and nutritional food quality. UPFs were analyzed based on fat sour ces, type of food and correlations between the different components among the studied food categories to establish a possible trend related to the relationship with fat source, sugars, salt, and calorie content. 3.2 MATERIALS AND METHODS 3.2.1 Materials, Chemicals , and Reagents Methanol was from Sigma - Aldrich (St. Louis, MO). Chloroform was obtained from Omni Solv (Burlington, MA), hexane was purchased from VWR BDH Chemicals (Batavia, IL), 1 - butanol and potassium chloride (KCl) from J. T. Baker (Allentown, PA) and di ethyl ether was purchased from Fisher Chemical (Pittsburgh, PA). Sodium sulfate anhydrous (Na 2 SO 4 ) and sodium chloride (NaCl) were also purchased from VWR BDH Chemicals. Supelco 37 FAME standard mixture was purchased from Sigma Aldrich (St Louis, MO). 3.2.2 Sample C ollection Composite foods and food products of different categories of UPFs were collected from retail stores, supermarkets, food chains, restaurants, and takeaway in Lansing area (Michigan, USA) between February 2018 and October 2019. Different b rands and different 50 retailers for the same type of food were acquired to achieve a representative sample. A complete list of the food meals, their respective test code, and group is provided in Appendix C . Meals were classified in two main groups: Fast Foo ds (FFs) and Ready to Eat (RTE), where 23 FF meals were purchased from the eight most popular franchisees in the state of Michigan covering more than 75% of the national market (Dunford et al., 2017; Powell et al., 2019; Rwithley, 2019; Tran et al., 2019) . In addition, f ood items and meals were grouped in products (D) , meat and poultry (MP), seafood (S), baby food (BF). Additional food items that did not fit in any of the previous categories, such as potato - products (potato crisps with and without added flavors, French fries from restaurants and takeaway, frozen potato es pre - fried and fried, and homemade French fries), pasta, salad dressings, and popcorn (sweet or salty) were grouped as other products (O). Once the UPF arrived at the laboratory, an excel form e; (2) price; (3) place, date, and time of collection; (4) type of food (RTE or FF); (5) nutritional declaration (energy, fat, saturated fatty acids, carbohydrates, sugars, fiber, protein and salt); (6) portion size; (7) list of ingredients; (8) expiring d ate; and (9) other relevant information. 3.2.3 Sample P reparation FF meals were purchased from each individual franchise and brought to the laboratory for immediate analysis. RTE meals were purchased from different local supermarket stores, and immediately brought to the laboratory. Storage conditions were followed according to the label instructions (fresh foods were kept in a fridge at 4°C and frozen meals were kept at - 20°C or the temperature indicated in the label). All meals were analyzed be fore the expiration date. When further cooking procedures were required, items 51 is study. All the samples were homogenized using an Ultra - Turrax® (Tekmar TP 18/10S1 Cincinnati, OH) at least for 3 min at 5000 rpm, split and stored accordingly, depending on the food matrix. 3.2.4 Lipid Extraction Lipid fraction was extracted according to the Folch cold extraction method (Folch, Lees, & Sloane Stanley, 1957) with some modifications depending the food matrix. Thirty grams of sample were m inced and placed in a 500 mL glass bottle with screwcap where 200 mL of a chloroform:methanol solution (1:1, v/v) was added. Sample was mixed for 15 min at 300 rpm. Homogenization was performed using an Ultra - Turrax for 3 minutes. The bottle was kept in an oven at 60°C for 20 min before adding 100 mL chloroform. After 2 min of vortex mixing the sample, the content of the bottle was filtered. The filtrate was mixed thoroughly with a 100 mL of 1 M KCl solution, and left overnight at 4 °C. Then, the lower phas e containing lipids was collected and dried at 60°C with a multi - vacuum solvent evaporator (Organomation S - EVAP - RB, Berlin MA) at 25 inches of Hg. Total fat content was determined gravimetrically. 3.2.5 Fatty A cid M ethyl E sters (FAME) Fatty acid methyl esters (FAME) were prepared according to the transesterification (Y. S. Chen et al., 2019) of the methylated sample was injected into a gas chromatography (GC 2010 Shimadzu, Kyoto Japan) equipped with a DB - was set as follows: 120 ° C to 200°C with a rate of 3°C/m in, then from 200 ° C to 240 ° C with a rate of 2 °C/min and held for 2 min. Injector and detector were both set at 250°C. H 2 52 was used as carrier gas at 1mL/min and split ratio 5.0. Data acquisition was done by Lab solutions software (Shimadzu, Kyoto, Japan) and peaks areas were identified by comparing their retention times to the pure standards. Standard curves of FAME 37 mixture were built with different concentrations from 65 to 500 ug/mL. Fatty acid contents were reported as the weight percentage of total fatty acid detected (% w/w) per g of fat. SFA = C8:0 + C10:0 + C11:0 + C12:0 + C13:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0 + C21:0 + C22:0 + C23:0 + C24:0); cis - 9 + C15:1, cis - 10 + C16:1, cis - 9 + C17:1, cis - 10 + C18:1, cis - 9 + C20:1, cis - 11 + C22:1, cis - 13 + C24:1, cis - - 9,12 + C18:3, cis - 6,9,12 + C18:3, cis - 9,12,15 + C20:2, cis - 11,14 + C20:3, cis - 8,11,14 + C20:3, cis - 11,14,17 + C20:4, cis - 5,8,11,14 + C22:2, cis - 13,16 + C20:5, cis - 5,8,11,14,17 + C22:6, cis - - 9 + C18:2, trans - 9,12). 3.2.6 Statistical A nalys is Descriptive statistics were calculated overall and by category. Both mean, and confidence interval (95%) were reported. Since the data did not follow a normal distribution, when comparing RTE vs FF items, a Matt - Whitney U - test was performed, at p < 0.05 significance level. Statistical differences between food categories were evaluated by means of the non - parametric Kruskal - Wallis ANOVA by Ranks test, followed by post - hoc comparisons of mean ranks of all pairs of groups. Before statistical assessment, FAM E percentages were arcsine square root transformed (Olson, 1976) . All the statistical analysis were computed using SPSS v.26 (IBM). 53 3.3 RESULTS AND DISCUSSION 3.3.1 Total Fat, Sugar and Sodium in UPFs The overall total fat ranges from 0.60 to 87.62 g/100 g of product with the dairy category (n=11) being the group with the highest fat content followed by the eggs and egg's derivatives category (n=2) with up to 77 g/100 g of product (Table 3 - 1). Figure 3 - 1 ( A - D ) shows a boxplot distribution of fat and FAME groups within each category. UPFs meals and food products were grouped based on their fat source as describe above. Some food products like edible fats (olive, avocado, canola, etc.) were not in cluded since they are considered foods themselves with no additional components. Due to the large number of UPFs in this study, total fat will be primarily discussed for each food category, detailing it for individual meals only when a distinct result is s tatistically significant. FF and RTE meals are known to be served in large portions, containing high levels of saturated fat, and added sugars (Harris et al., 2013; Rosenheck, 2008). Most of these meals are prepared with several ingredients such as oils, e ggs, high fat meats - all of them containing high fat content themselves - resulting in a meal with both high fat and high caloric content. 3.3.2 Fatty A cid Pr ofile by F ood C ategory Fatty acid composition of RTE and FF meals is shown in Appendix D. Thirty - five fatty acids were identified and quantified. Short chain fatty acids (SC - FA) were not reported since C4 and C6 co - eluded with the solvent. Also, dihomo - - linolenic acid (C20:3n - 3 , DGLA) and eicosatrienoic acid (C20:3n - 6) were not chromatographically resolved for some samples, therefore their area values were merged and reported as C20:3n - 3 + C20:3n - 6. Tables 3 - 2 to 3 - 4 show the percentage of saturated fatty acids (SFA), monounsatu rated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), respectively. 54 Table 3 - 1 : Food ID used in this study and unpublished results of total fat, sugar, sodium, and calories per serving found in the differe nt UPFs. Category Food ID Total Fat (g/100 g sample) Total Fat Nutritional label ** (g/100 g sample) Serving Size Total Fat (g) pe r serving size Sugar (g) per serving size Sodium (mg) per serving Calories per serving Ready to Eat Dairy D1 - RTE 17.67 ± 2.45 18.40 19.00 g 3.50 1.00 280 60 D2 - RTE 26.92 ± 8.47 33.00 21.00 g 6.93 0 180 110 D3 - RTE 49.71 ± 2.56 50.00 1 tbsp (14.15 g) 7.08 NR 100 60 D4 - RTE 87.62 ± 1.91 78.57 14.00 g 11.00 0 90 100 D5 - RTE 10.27 ± 0.38 10.00 30.00 mL (28.3 g) 2.83 1 25 40 D6 - RTE 24.16 ± 6.98 28.00 28.00 g 7.84 1 240 60 D7 - RTE 42.82 ± 1.68 35.00 28.00 g 9.80 2 95 90 D8 - RTE 12.78 ± 3.04 23.40 0.50 cup (65.00 g) 15.21 14 40 130 D9 - RTE 1.60 ± 0.31 3.03 1 container (99.00g) 3.00 20 90 150 D10 - RTE 2.14 ± 0.54 0.96 414.03 g 0.60 37 230 250 D11 - RTE 12.56 ± 0.38 0.69 1 scoop (9.00 g) 0.062 NR 0.32 44 Meat & Poultry MP1 - RTE 23.45 ± 5.00 20.00 38.00 g 7.60 < 1 260 90 MP2 - RTE 17.84 ± 2.65 26.00 32.00 g 8.30 0 260 50,000 MP3 - RTE 0.60 ± 0.071 0.65 240.00 mL (226.40 g) 1.50 3 870 170 MP4 - RTE 3.64 ± 0.018 3.30 1 can (236.59 g) 7.80 5 870 250 MP5 - RTE 3.53 ± 0.55 5.65 11.00 oz (227.00 g) 12.80 5 500 270 MP6 - RTE 1.37 ± 0.31 1.76 0.5 cup (113.40 g) 2.00 0 640 60 MP7 - RTE 3.61 ± 0.013 0.61 0.5 cup (240.00 g) 1.50 1 600 70 MP8 - RTE 2.91 ± 0.53 2.87 1 cup (249.00 g) 7.15 6 800 200 MP9 - RTE 4.61 ± 0.097 4.28 1 cup (257.00 g) 11.00 8 800 260 Seafood S1 - RTE 3.62 ± 0.25 3.38 18.80 oz (532.97 g) 18.01 1 890 180 Derivatives E1 - RTE 77.68 ± 1.95 75.00 1 Tbsp (13 g) 9.75 NR 90 90 E2 - RTE 14.64 ± 1.02 13.91 ½ cup (115.00 g) 16.00 10 330 380 Baby Food BF1 - RTE 1.80 ± 0.15 1.41 71.00 g 1.00 0 35 50 BF2 - RTE 7.28 ± 0.19 4.40 4.00 oz (113.40 g) 4.99 NS 40 90 55 Table 3 - 1 Category Food ID Total Fat (g/100 g sample) Total Fat Nutritional label ** (g/100 g sample) Serving Size Total Fat (g) per serving size Sugar (g) per serving size Sodium (mg) per serving Calories per serving Ready to Eat Baby Food BF3 - RTE 2.99 ± 0.06 4.40 4.00 oz (113.40 g) 4.99 3 30 70 BF4 - RTE 3.25 ± 0.23 4.40 4.00 oz (113.40 g) 4.99 3 40 70 BF5 - RTE 3.23 ± 0.14 4.40 4.00 oz (113.40 g) 4.99 3 45 80 BF6 - RTE 2.12 ± 0.60 1.56 128.00 g 2.00 5 260 120 BF7 - RTE 3.09 ± 0.16 4.40 4.00 oz (113.40 g) 4.99 5 20 80 BF8 - RTE 6.00 ± 0.14 4.90 71.00 g 3.48 0 20 50 BF9 - RTE 2.79 ± 0.44 4.40 4.00 oz (113.40 g) 4.99 11 50 100 BF10 - RTE 4.63 ± 0.48 4.40 4.00 oz (113.40 g) 4.99 4 40 80 BF11 - RTE 3.89 ± 0.23 4.40 4.00 oz (113.40 g) 4.99 4 80 120 BF12 - RTE 2.90 ± 0.38 4.40 4.00 oz (113.40 g) 4.99 6 75 90 BF13 - RTE 1.39 ± 0.25 2.35 85.00 g 2.00 1 170 80 Other O1 - RTE 19.97 ± 1.85 26.60 2 tbsp (30.00 g) 7.98 0 290 140 O2 - RTE 45.11 ± 6.32 50.00 2 tbsp (30.00g) 15.00 1 230 140 O3 - RTE 8.70 ± 5.00 5.00 1 package (58.00 g) 2.90 6 570 250 O4 - RTE 3.65 ± 1.51 5.00 2.5 oz (70.90 g) 3.55 4 500 220 Fast Food Meat & Poultry MP10 - FF 33.13 ± 5.12 10.00 95.00 g 9.50 NR 510 250 M11 - FF 19.52 ± 1.46 20.00 4 pieces (64.00 g) 12.80 NR 330 170 MP12 - FF 23.59 ± 3.40 14.00 119.00 g 16.66 NR 720 300 MP13 - FF 9.83 ± 0.45 11.40 1 taco (102.00 g) 11.63 1 306 269 MP14 - FF 11.06 ± 2.09 7.60 1 quesadilla (170.00 g) 12.92 0 310 180 MP15 - FF 9.95 ± 0.67 9.90 1 burrito (140.00 g) 13.86 1 1650 640 MP16 - FF 12.48 ± 0.30 12.21 1 piece (75.00 g) 9.16 0 430 130 56 Table 3 - 1 Category Food ID Total Fat (g/100 g sample) Total Fat Nutritional label ** (g/100 g sample) Serving Size Total Fat (g) per serving size Sugar (g) per serving size Sodium (mg) per serving Calories per serving Fast Food Meat & Poultry MP17 - FF 19.39 ± 2.55 18.60 1 piece (60.00 g) 11.16 0 380 130 MP18 - FF 6.26 ± 1.21 4.58 5.40 oz (153.09 g) 7.01 7.00 520 150 MP19 - FF 8.19 ± 0.78 10.80 5.70 oz (161.69 g) 17.46 19.00 820 490 MP20 - FF 14.23 ± 2.82 10.50 1 sandwich (187.00 g) 19.64 9.00 680 320 MP21 - FF 7.12 ± 1.18 6.51 1 sandwich (71.28 g) 4.64 3 2370 810 MP22 - FF 10.74 ± 4.18 8.79 1 sandwich (94.61 g) 8.32 2 1940 850 MP23 FF*** 12.59 ± 1.35 11.50 1 slice (123.00 g) 14.15 3 950 370 MP24 - FF 10.96 ± 2.70 11.50 1 slice (79.00 g) 9.09 2.00 740 380 Seafood S2 - FF 22.82 ± 1.07 14.08 1 sandwich (131.00 g) 18.44 NR 580 380 S3 - FF 21.70 ± 0.15 13.10 5.00 oz (141.75 g) 18.57 14.00 440 360 Other O5 - FF 13.92 ± 1.58 20.00 1 medium serving (117.00 g) 23.40 NR 260 320 O6 - FF 16.27 ± 2.86 16.00 1 biscuit (76.00 g) 12.16 NR 810 260 O7 - FF 6.29 ± 1.34 6.00 3 hotcakes (149.00 g) 8.94 NR 550 580 O8 - FF 18.16 ± 0.75 17.30 1 biscuit (49.00 g) 8.48 1 520 180 O9 - FF 15.96 ± 0.21 15.00 1 order (34.99 g) 5.25 0 1100 320 O10 - FF 1.46 ± 0.52 3.11 1 order (16.85 g) 0.52 0 520 130 **Information found in the USDA Food Database. www.fdc.nal./usda.gov/fdc - app.html#/ ***Information found in Menuwithprice.com. Not available in neither USDA Food NR = not reported NS = not a significant source of the nutrient 57 Figure 3 - 1 : Unpublished results of (A) fat content vs. food categories, (B) SFA vs. food categories, (C) MUFA vs. food categories, (D) PUFA vs. food categories, (E) calories per servings vs. food categories, and (F) sodium vs. food categories 58 Figure 3 - 1 (B - D) depicts the overall distribution of FAME categories (SFA, MUFA, PUFA) according to the food source. No statistically significant differences were found between FF and RTE groups. The meat and poultry category (n=24) which includes beef, chicken, pork, tur key, and combinations of these meats, had the third largest amount of total fat (values ranging from 0.60 to 33.13 g/100 g of product). The majority of UPFs showed a higher fat content compared to the values reported either in their nutritional label or we bsite. Higher fat contents were observed in several dairy products containing butter as part of its use in their preparation process. Butter is the only processed food included in this study due to the great use in the manufacturing of several UPFs. 3.3.3 Satur ated F atty A cids (SFA) Saturated fatty acid was the overall second major content of fatty acids between both FF and RTE meals with 35.57%, just behind the monounsaturated fatty acids (37.82%). The total SFA content was significantly different between RTE and FF with 40.88 and 29.88 g/100g of product, respectively (Table 3 - 2) . C16:00 was the predominant SFA in both RTE and FF meals with a mean of 21.74 g/100 g, followed by C18:00 with 7.61 g/100g. Regarding the length of the saturated chain in t he fatty acid, long chain fatty acid (LC - SFA) accounted for more than 90% of the total SFA Content in comparison with 5.3% of the medium chain saturated fatty acid (MC - SFA). Dietary triacylglycerols are derived principally from two sources: animal fats and vegetable oils. Animal fats contain a high proportion of SFA as for butter, which SFA content mostly consists in 4 SFAs ( C12:00, C14:00, C16:00, and C18:00 ) (Bobe, Hammond, Freeman, Lindberg, & Beitz, 2003; Ledoux et al., 2005; Lopes, Cañedo, Oliveira, & Alcantara, 2018) . On the other hand, vegetable oils have a higher 59 proportion of unsaturated fatty aci ds. Since LCFA are hydrophobic nutrients, their intestinal absorption is a complex process. Fatty - acid chain length and unsaturation number influence fat absorption. In hu man diet, approximately 95% of dietary lipids are triacylglycerols (TAG), mainly composed of long - chain fatty acids (LCFA, number of carbons >16). Medium - chain fatty acids (MCFA) are better absorbed than LCFA because they can be solubilized in the aqueous phase of the intestinal contents, absorbed bound to albumin, and transported to the liver by the portal vein (Wang et al., 2013) . Further , absor ption in the stomach occurs after the hydrolysis of medium - chain triglycerides (MCT) by gastric lipase, enhancing their solubilization in the intestine, where they are absorbed bound to albumin and transported to the liver via the portal vein (Niot et al., 2009) . LCFA exert basic functions in the cell as membrane components, metabolic fuel, precursors of lipid mediators, regulators of ion channel s and modulators of gene expression (Linder & Deschenes, 2007) . They participate in several post - translational protein modifications (e.g., palmitoylation) affecting their cellular functions. Moreover, dietary lipid fecal loss remains below 5% (w/w), still with a high fat intake in healthy individuals (Ram rez, Amate, & Gil, 2001) . Hence, lipid uptake is not a rate - limiting step for the intestinal fat absorption, and more attention should be paid to the meals size portion and fat source to maintain a heal thy diet. Interestingly, albeit not surprising considering the lipid needs for infants, infant formula has the overall highest SFA content from all UPFs with 56%. SFA content in infant formulas containing dairy as their major source of fat is significantly higher in comparison with those having plant oils as their main source ( C. Sun, Wei, Su, Zou, & Wang, 2018) . 60 Table 3 - 2 : Unpublished results of the saturated fatty acid composition in RTE and FF meals SFA Total Ready - To - Eat Fast - Food Mann - Whitney U - test C8:0 1.18 (0.08 - 1.59) 1.43 (0.08 - 2.03) 0.64 (0.49 - 0.80) C10:0 1.74 (1.49 - 2.00) 2.01 (0.17 - 2.36) 1.22 (1.03 - 1.41) * C11:0 0.06 (0.05 - 0.08) 0.07 (0.06 - 0.09) 0.04 (0.02 - 0.05) *** C12:0 2.05 (1.63 - 2.46) 2.04 (1.57 - 2.51) 2.06 (1.22 - 2.89) C14:0 3.54 (2.99 - 4.09) 4.22 (3.43 - 5.02) 2.36 (1.81 - 2.91) ** C15:0 0.42 (0.36 - 0.49) 0.51 (0.42 - 0.06) 0.28 (0.21 - 0.34) * C16:0 21.74 (20.52 - 22.96) 23.96 (22.34 - 25.57) 18.09 (16.57 - 19.60) **** C17:0 0.35 (0.31 - 0.39) 0.39 (0.34 - 0.44) 0.29 (0.22 - 0.35) ** C18:0 7.61 (7.10 - 8.12) 8.41 (7.69 - 9.13) 6.29 (5.76 - 6.82) *** C20:0 0.29 (0.26 - 0.32) 0.24 (0.21 - 0.28) 0.35 (0.31 - 0.40) *** C21:0 0.30 (0.24 - 0.35) 0.31 (0.22 - 0.40) 0.27 (0.23 - 0.32) C22:0 0.12 (0.08 - 0.15) 0.12 (0.07 - 0.17) 0.11 (0.06 - 0.17) C23:0 0.05 (0.03 - 0.08) 0.03 (0.02 - 0.04) 0.07 (0.03 - 0.10) *** C24:0 0.22 (0.13 - 0.32) 0.21 (0.06 - 0.35) 0.24 (0.10 - 0.37) *** SFA 35.57 (33.01 - 38.13) 40.88 (37.48 - 44.29) 29.88 (27.17 - 32.60) **** MC - SFA 3.66 (2.98 - 4.34) 3.94 (3.05 - 4.82) 3.08 (2.06 - 4.10) LC - SFA 33.66 (31.59 - 35.72) 37.31 (34.48 - 40.15) 27.61 (25.36 - 29.86) **** SFA: Sum of saturated fatty acid, MC - FA : sum of medium chain saturated fatty acid, LC - FA long chain saturated fatty acid . Even though the majority of SFA in both RTE and FF meals were straight chain even - numbered homologues (i.e., C12:00, C14:00, C16 :00, etc.), C15:00, C17:00, and C23:00 were also detected in UPFs, being C15:00 the only statistically significant. Dairy foods, comprised of RTE items, contain the highest amount of C15:00 with 1.02 g/100g of product followed by the baby foods category. A ll type of cheeses (American, Cheddar, and Swiss) showed a high SFA content which agrees with literature (Manuelian, Currò, Penasa, Cassandro, & De Marchi, 2017) ; except for the cream cheese, which l iterature values were 61 not available to confirm their high SFA content. Mac & Cheese (boiled, prepared) showed a higher fat content than the Mac & Cheese prepared by microwave heating. These results demonstrate that cooking techniques and preparation are cr itical parameters that should be considered when evaluating nutritional quality of foods. Total fat content for butter and ice cream agreed with previous studies (Nielsen, 1996a; Tavella et al., 2000) and the USDA food database (USDA, 2018) . contained 30.7% SFA, which was confirmed with their ref erence values in the USDA database (Huang et al., 2017; USDA, 2019b, 2019c) . Palmitic acid (C16:0), lauric acid (C12:0), stearic acid (C18:0), and myristic acid (C14:0) were the four leading FA between the RTE and FF meals are the UPFs with a major contribution of fat accumulation in the human body in the Western diet (Mohiuddin, 2019) , which is directly associated to different chronic diseases such as obesity, diabetes, hypertension , atherosclerosis , among others (Shori, Albaik, & Bokh ari, 2017) . Therefore , the quantification of total fat in RTE and FF meals is important to evaluate the actual dietary fat intake of adults and children who consume large amount of UPFs. 3.3.4 Monounsaturated F atty A cids (MUFA) MUFA was the group with the highest content among all UPFs. FF values ranged from 40 to 45 (g/100 product) (Table 3 - 3 ) while RTE values range from 34 to 38 (g/100 product). It is worth mentioning that RTE meals contain higher percentages of SFA (40.88 g/100 of product) than the overa ll percentage of MUFA among UPFs. Conversely, the FF group contains the highest percentage of MUFA than the overall amount of both SFA and MUFA in 62 all UPFs. Furthermore, the oleic acid (C18:1, cis - 9, OA) was the dominant fatty acid in all UPFs with more th an 90% of the total MUFA. OA not only provides energy but also reduces the melting point of triacylglicerides ( Ram rez, Amate, & Gil, 2001 ). Baby foods and meat & poultry were the categories with the higher content of OA, followed by seafood. Table 3 - 3 : Unpublished results of monounsaturated and polyunsaturated fatty acid composition in RTE and FF meals MUFA Total Ready - To - Eat Fast - Food Mann - Whitney U - test C14:1 0.55 (0.48 - s0.62) 0.59 (0.52 - 0.67) 0.46 (0.32 - 0.60) *** C15:1 0.18 (0.12 - 0.25) 0.18 (0.12 - 0.25) ND - C16:1 1.76 (1.56 - 1.96) 1.83 (1.57 - 2.09) 1.63 (1.31 - 1.95) C17:1 0.26 (0.22 - 0.31) 0.26 (0.20 - 0.31) 0.28 (0.18 - 0.38) tC18:1 1.23 (0.77 - 1.70) 2.14 (0.53 - 3.75) 0.93 (0.59 - 1.27) C18:1 n - 9 36.09 (34.65 - 37.53) 33.64 (31.88 - 35.39) 40.15 (37.92 - 42.38) **** C20:1 0.40 (0.36 - 0.44) 0.33 (0.28 - 0.39) 0.49 (0.42 - 0.55) **** C22:1 0.28 (0.20 - 0.36) 0.19 (0.15 - 0.24) 0.34 (0.21 - 0.47) *** C24:1 0.19 (0.09 - 0.28) 0.14 (0.05 - 0.23) 0.27 (0.02 - 0.57) *** MUFA 37.82 (36.01 - 39.63) 36.57 (34.64 - 38.50) 43.17 (40.73 - 45.61) **** PUFA Total Ready - To - Eat Fast - Food Mann - Whitney U - test tC18:2 0.35 (0.30 - 0.40) 0.39 (0.31 - 0.46) 0.31 (0.23 - 0.39) C18:2 n - 6 20.88 (18.81 - 22.95) 19.59 (16.88 - 22.29) 23.02 (19.82 - 26.22) * C18:3n - 6 0.10 - (0.08 - 0.12) 0.11 (0.08 - 0.14) 0.09 (0.07 - 0.10) C18:3n - 3 2.70 (2.37 - 3.03) 2.7 (2.21 - 3.14) 2.75 (2.31 - 3.19) C20:3n3 + C20:3n6 0.12 (0.10 - 0.13) 0.12 (0.10 - 0.15) 0.11 (0.10 - 0.13) C20:4n - 6 0.11 (0.05 - 0.17) ND 0.11 (0.05 - 0.17) C20:5n - 3 0.27 (0.16 - 0.39) 0.21 (0.12 - 0.30) 0.32 (0.12 - 0.51) *** C22:2 n - 6 0.06 (0.00 - 0.13) 0.02 (0.01 - 0.04) 0.13 ( - 0.13 - 0.40) **** C22:6 n - 3 0.05 (0.04 - 0.07) 0.05 (0.03 - 0.07) 0.06 (0.04 - 0.07) *** PUFA n - 3 0.17 (0.12 - 0.21) 0.16 (0.10 - 0.21) 0.18 (0.09 - 0.28) PUFA n - 6 23.88 (21.54 - 26.21) 22.55 (19.46 - 25.63) 26.07 (22.51 - 29.63) * PUFA 23.62 (21.29 - 25.95) 22.85 (19.79 - 25.91) 26.95 (23.43 - 30.46) * PUFA: Sum of polyunsaturated fatty acid, PUFA n - 6 sum of omega 6 polyunsaturated fatty acid, PUFA n - 3: sum of omega 3 polyunsaturated fatty acid. MUFA: sum of monounsaturated fatty acid, MUFA are reported in %. 63 The high level of OA on these meals may be due to the use of soybean and high oleic sunflower oil in their recipes. Palmitoleic acid (C16:1), oleic acid (C18:1, cis - 9), and elaidic acid (C18:1, trans - 9) were the second, third, and fourth most abundant MUFA in the meat & poultry category, respectively. Values are aligned with those reported previously (Haak, De S met, Fremaut, Walleghem, & Raes, 2008; Karakok, OzogulL, Saler, & Ozogul, 2010) and with the information reported in their nutritional labels. Even though the majority of the MUFA reported in the USDA database were detected in our samples, some of them were present in small quantities in the USDA database and not detected in our samples and vice versa. Moreover, French fries from two different f ranchises showed higher MUFA percentage compared to the other FA groups. Twelve UPFs containing beef as their major component showed the highest fat content among the meat & poultry category. (See Table 3 - 4 ) This abundant presence of fat in beef is also ob served in other food items such as roasted rib (style: large end) which contains 26 g per serving size (3 oz ) (USDA, 2019a) , and in ground beef which can contain a maximum 30% fat allowed by the USDA (USDA, 2016) . Previous studies in beef have reported SFA and MUFA as their dominating FAME group (Karakok et al., 2010; Wood, 1996) . Beef with vegetables from an FF restaurant, reported a saturated fat content of 21.4% in their website, which is slightly lower than our 26.94% of SFA, this confirms that our res ults could be more accurate. Baby foods are RTE meals with the highest fat and MUFA contents, which are required to chicken broth and canola oil. MUFA was dominated by oleic acid (C18:1, cis - 9), palmitoleic acid (C16:1), and 11 - eicosenoic acid (C20:1). PUFA in baby foods was led by linolenic acid, LA (C18:2cis), and - 64 linolenic acid (C18:2 n - 6), GLA (C18:3n6). Lastly, 3 trans FAs were detected in our samples: elaidi c acid (C18:1,trans - 9), palmitelaidic acid (C16:1trans) and rumenic acid (C18:2trans), with elaidic acid (C18:1,trans - 9) being the trans FA with the highest amount. 3.3.5 Polyunsaturated F atty A cids (PUFA) Dietary fat is directly related with the use of vegetables oils and other ingredients such a fish, avocado, among others, which contain high PUFA levels. PUFA was the third most abundant group in all UPFs with no significant differences between FF and RTE. Linoleic acid (C18:2 n - - linolenic acid (C18:3 n - 3, LNA). The has the highest content of LA followed by seafood. These results agree with literature (Bemrah, Sirot, Leblanc, & Volatier, 2009) Simopoulos & Salem, 1992). The main ingredient of macaroni salad is mayonnaise, and both have similar fatty acid profile in agreement with those reported in literature (Tavella et al., 2000) . Therefore , PUFA was also the predominant group in macaroni salad and traces presence of trans fatty acid (18:2, trans - 9,12) was detected just in mayonnaise. Less than 1% of the tot al fat contained trans fatty acids. Results agree with federal regulations about the total content of trans fatty acid in foodstuff (Gonçalves Albuquerque, Santos, Silva, Oliveira, & Costa, 2018) . Higher PUFA content was found in salad dressing, where results agree with previous studies that reported similar values (Jacobsen, 2015; Let, Jacobsen, & Meyer, 2007) . It is worth to mention that the effect of cooking method on FA profiles, for example Mac & Cheese meals were cooked using 2 different cooking methods (boiled and microwaved, Table 3 - 1). No reference values were found both types of cooking methods emp loyed in this study, however the major ingredient on these meals is cheddar 65 Table 3 - 4 : Unpublished results of the fatty acid profile of UPFs by foo category (mean and range) SFA Meat & Poultry (n=24) Dairy (n=9) Eggs & Egg products (n= 2) Seafood (n=3) Baby Foods (n=13) Others (n=12) p - value ¶ C8:0 0.42 (0.32 - 0.52) 1.05 (0.97 - 1.12) 0.15 (0.06 - 0.24) ND 2.43 (0.07 - 4.79) 2.05 (0.04 - 3.69) **** C10:0 1.19 (0.96 - 0.14) 2.62 (2.34 - 2.90) 0.05 (0.04 - 0.07) d 0.79 ( - 0.13 - 0.17) 2.50 (1.83 - 3.16) 1.55 (0.59 - 2.51) **** C11:0 0.04 (0.02 - 0.05) 0.07 (0.06 - 0.09) ND ND 0.07 (0.05 - 0.10) ND *** C12:0 1.02 (0.77 - 1.28) 4.36 (3.38 - 5.35) 0.03 (0.01 - 0.04) 1.03 ( - 0.07 - 0.21) 1.25 (0.72 - 0.18) 2.93 (1.51 - 4.34) **** C14:0 2.61 (2.10 - 3.13) 1.06 (0.98 - 1.15) 0.09 (0.08 - 0.10) 1.61 (0.10 - 0.31) 3.29 (2.10 - 4.49) 1.52 (0.97 - 2.08) **** C15:0 0.37 (0.31 - 0.43) 1.02 (0.86 - 1.18) ND 0.18 (0.05 - 0.30) 0.43 (0.29 - 0.58) 0.11 (0.06 - 0.15) **** C16:0 20.77 (19.67 - 21.87) 34.29 (33.57 - 35.01) 10.99 (10.92 - 11.07) 13.63 (12.02 - 15.24) 20.32 (17.76 - 22.87) 19.80 (16.31 - 23.30) **** C17:0 0.44 (0.36 - 0.51) 0.50 (0.42 - 0.57) 0.09 (0.08 - 0.10) 0.19 (0.14 - 0.24) 0.33 (0.27 - 0.40) 0.12 (0.10 - 0.14) **** C18:0 8.43 (7.60 - 9.27) 11.37 (10.19 - 12.55) 4.08 (4.01 - 4.15) 4.99 (4.59 - 5.39) 6.78 (5.83 - 7.74) 5.32 (4.68 - 5.96) **** C20:0 0.28 (0.23 - 0.34) 0.17 (0.12 - 0.22) 0.29 (0.27 - 0.31) 0.37 (0.33 - 0.41) 0.29 (0.23 - 0.36) 0.35 (0.30 - 0.39) ** C21:0 0.11 (0.05 - 0.17) ND ND ND ND ND - C22:0 0.39 (0.19 - 0.59) 0.17 (0.16 - 0.19) 0.29 (0.24 - 0.34) 0.30 (0.23 - 0.37) 0.17 (0.11 - 0.23) 0.22 (0.18 - 0.27) - C23:0 0.02 (0.02 - 0.03) 0.08 (0.02 - 0.14) ND ND 0.03 ( - 0.08 - 0.14) 0.04 (0.02 - 0.05) - C24:0 0.31 (0.09 - 0.54) 0.13 (0.11 - 0.14) ND 0.11 (0.11 - 0.12) 0.25 (0.02 - 0.47) 0.12 (0.10 - 0.15) - SFA 34.33 (31.13 - 36.53) 66.06 (64.23 - 67.89) 16.07 (15.91 - 16.22) 22.23 (17.99 - 26.46) 33.64 (28.07 - 39.21) 30.71 (25.63 - 35.79) **** MUFA Meat & Poultry Dairy Eggs & Egg products Seafood Baby Foods Others p - value C14:1 0.49 (0.39 - 0.60) 0.94 (0.89 - 0.99) ND 0.48 ( - 0.45 - 1.43) 0.53 (0.38 - 0.67) 0.17 (0.07 - 0.27) **** C15:1 ND ND ND ND 0.18 (0.12 - 0.25) ND - C16:1 2.49 (2.19 - 2.79) 1.33 (1.06 - 1.59) 0.12 (0.11 - 0.13) 0.75 (0.15 - 1.36) 2.25 (1.78 - 2.72) 0.43 (0.28 - 0.58) **** C17:1 0.37 (0.27 - 0.48) 0.27 (0.18 - 0.36) 0.04 (0.02 - 0.07) 0.15 (0.03 - 0.27) 0.26 (0.19 - 0.34) 0.06 (0.05 - 0.07) **** tC18:1 1.44 (0.82 - 2.06) ND ND ND 0.14 ( - 0.09 - 0.36) 1.04 (0.19 - 1.89) - C18:1 n - 9 39.83 (38.26 - 41.40) 25.86 (24.89 - 26.83) 21.41 (21.38 - 21.44) 30.21 (24.22 - 36.19) 40.30 (36.99 - 43.61) 33.58 (29.69 - 37.47) **** C20:1 0.49 (0.43 - 0.55) 0.09 (0.07 - 0.10) 0.15 (0.14 - 0.16) 0.29 (0.16 - 0.43) 0.49 (0.39 - 0.59) 0.32 (0.23 - 0.41) **** C22:1 0.06 (0.02 - 0.09) ND ND 0.21 (0.16 - 0.26) 0.12 (0.07 - 0.18) ND - C24:1 0.03 (0.02 - 0.07) 0.17 (0.13 - 0.20) ND 0.52 (0.34 - 0.71) 0. 13 (0.07 - 0.26) ND *** 66 Table 3 - 4 MUFA Meat & Poultry Dairy Eggs & Egg products Seafood Baby Foods Others p - value MUFA 44.00 (42.29 - 45.71) 28.31 (27.53 - 28.82) 21.73 (21.67 - 29.10) 31.37 (25.02 - 37.71) 43.71 (40.25 - 47.17) 34.67 (30.65 - 38.69) **** PUFA Meat & Poultry Dairy Eggs & Egg products Seafood Baby Foods Others p - value tC18:2 0.34 (0.27 - 0.41) 0.53 (0.42 - 0.64) 0.04 (0.03 - 0.05) ND 0.40 (0.30 - 0.49) 0.14 (0.07 - 0.29) **** C18:2 n - 6 18.42 (16.33 - 20.51) 4.40 (3.06 - 5.74) 54.95 (54.81 - 55.09) 40.57 (33.29 - 47.84) 18.86 (15.84 - 21.87) 31.22 (25.86 (36.58) **** C18:3n - 6 0.12 (0.10 - 0.15) 0.04 (0.04 - 0.05) ND ND 0.10 (0.05 - 0.15) 0.10 (0.06 - 0.13) - C18:3n - 3 2.12 (1.81 - 2.44) 0.52 (0.43 - 0.62) 7.13 (7.08 - 7.19) 5.07 (4.20 - 5.93) 3.13 (2.38 - 3.89) 0.03886 (0.02912 - 0.0486) **** C20:2n - 6 0.77 (0.34 - 0.12) 0.01 (0.01 - 0.01) 0.04 (0.03 - 0.04) 0.20 ( - 0.27 - 0.67) 0.11 (0.06 - 0.16) 0.70 (0.11 - 1.51) *** C20:3n - 3 + C20:3n - 6 0.13 (0.12 - 0.14) 0.11 (0.09 - 0.13) 0.03 (0.02 - 0.04) 0.03 (0.02 - 0.05) 0.16 (0.09 - 0.23) 0.04 (0.01 - 0.06) *** C20:4n - 6 0.34 (0.28 - 0.39) 0.21 (0.18 - 0.24) 0.03 (0.02 - 0.04) 0.58 ( - 0.60 - 1.76) 0. 33 (0.21 - 0.46) 0.08 (0.05 - 0.10) - C20:5n - 3 0.42 (0.20 - 0.63) ND ND ND 0.15 (0.08 - 0.23) 0.07 (0.03 - 0.17) - C22:2n - 6 0.13 ( - 0.13 - 0.39) ND ND ND 0.02 ( - 0.01 - 0.04) 0.03 (0.03 - 0.06) - C22:6n - 3 0.05 (0.03 - 0.07) 0.02 (0.02 - 0.03) ND ND 0.06 (0.03 - 0.08) 0.09 (0.05 - 0.13) * PUFA 21.67 (19.37 - 23.98) 5.63 (4.21 - 7.04) 62.21 (62.02 - 62.40) 46.41 (38.80 - 54.02) 22.65 (19.28 - 26.02) 35.57 (29.35 - 41.79) ** SFA: sum of saturated fatty acid, sum of MUFA: monounsaturated fatty acid, Sum of PUFA: Polyunsaturated fatty acid. SFA, MUFA and PUFA are reported in %. ¶ Independent - samples Kruskal - Wallis test. 67 cheese which has been previously repor ted (Manuelian et al., 2017) and used for comparison purposes of this RTE. The microwaved sample showed a high SFA content while the boiled s ample showed PUFAs as its higher content, demonstrating the effect of temperature variability on food nutritional content. Seafoods are well known to be a great source of LCFA, especially omega 3 long - chain PUFA (Bemrah et al., 2009; Morais Júnior et al., 2017; Neff, Bhavsar, Braekevelt, & Arts, 2014; Oje et al., 2004; Sirot, 2008) cold water. These organisms are especially dependent on the physicochemical properties of the FA. This is due to the high number of double bonds in the molecule lowering the melting point of the compounds, which means that, even at low temperatures, biological structures significa nce for humans are cold - water fish, such as salmon, mackerel, herring, and tuna. The USDA recommends fish consumption of at least 8 oz (227 g) seafood per week in individuals two years old and olde r (Kantor, 2016) . Seafood can be found in food retail (56%) and restaurants (31%); being shrimp, clamps, salmon, cod, and Alaskan Pollock the most consumed seafood among US (Love et al., 2020). Contrary to what was expected, PUFA was the highest percentage in 2 of 3 of the seafood meals tested in this study. Some incongruencies were found after comparing certain PUFAs to previous literature values (Awogbemi, Onuh, & Inambao, 2019; Czech, Grela, & Ognik, 2015; Ekiz & Oz, 2019; Katan et al., 2020; Modzelewska - Pietrzak - . For example, in f ried shrimp from an FF restaurant, no C22:6 was detected opposed to what its reference values state; and instead of C18:3n3, we were able to identify just C18:3n6 68 (Czech et al., 2015) . Moreover, for the fish sandwich from another FF restaurant, PUFA was not the dominating FA group as stated in references because of the significant abundance of C18:2cis in our sample compared to the higher presence of C18:1cis reported by the USDA (USDA, 2020) . Results from RTE clam chowder soup are aligned with its reference values, however, a difference of 55.91% between PUFA and MUFA is reported in the USDA while our sample shows only a 1.05% difference. This suggest that either some PUFAs could be missing or that our sample contains higher amount of MUFAs. RTE soups like the clam chowder is a canned product that usually needs a reconstitution and warming steps before consumption. Differences during homemade preparation such as temperature changes, delay in wa rming, use of microwave vs boiling, etc. may increase the lipid oxidation process promoting autooxidation of lipid molecules which in turn, alters the overall fatty acid profile of the food item. Differences in the PUFA content were observed between biscu its from two FF franchises . For example, EPA was present in O8 - FF instead of ALA which was seen in O6 - FF ( APPENDIX C ). This could be the result of the use of milk as part of the ingredients in O8 - FF recipe which contains small amounts of EPA (USDA, 2019d) , in addition to cross con tamination of this FA using the same equipment for cooking procedure of different food meals which could contain EPA. Lastly, even though 4 trans FAs were reported by USDA, no trans FA was detected in O8 - FF. Overall, FA profile was similar to the one reported in the USDA database, a nd previous studies (Amrutha Kala, 2014; KFC, 2020; McDonald's, 2017; Rutkowska, , even though traces of trans FAs were reported by the USDA for French fries, none was detected in our samp le. It is noteworthy to mention that the variability in frying oils used as part of the French fries cooking procedure in these 2 FF restaurants, may result in the presence of elaidic acid in item O9 - FF, and its 69 absence in O5 - FF. As Smith and co - workers ex plained in their study (Song et al., 2015) the use of hydrogenated soybean oil for frying purposes caused the presence of high amounts of elaidic acid (C18:1, trans - 9), while the use of an animal - vegetable mix shortening (corn oil and beef flavor) in O5 - FF inhibited the formation of elaidic acid (C 18:1, trans - 9). 3.4 CONCLUSIONS This study provides an overview of the total fat and FAME profile composition of the most popular ultra - processed foods in the US Mid - West area in US. Convenience and palatability are the main reasons to consume UPFs. However, nutritional quality and dietary patterns are jeopardized by prices and popularity of UPF meals resulting in a public health issue which should be addressed. However, with respect to the sugar, salt, and saturated fat content, it was noticed that new challenges have arisen, especially focusing on the food industry and policy makers. Some key changes may have a great impact such as reformulation of menu items , including more vegetable and fruit options as side and/o desserts, reduction of serving size especially in beverages, sides, and desserts. The gradual reduction of the components previously mentioned in most of the food categories studied here should become a priority. It is possible to conclude t hat this subject remains present and new challenges are being pointed out by several national and international organizations . 70 CHAPTER 4 : C HOLESTEROL OXIDATION PRODUCTS ASSESMENT IN U LTRA - PROCESSED FOODS IN THE WESTERN DIET 4.1 INTRODUCTION Dietary patterns vary around the world depending on the countr and even regions within a same country . This variation is a result of differences in lifestyle and culture. Interestingly, these dietary patterns have changed over the past 60 years, cha nging the proportions of different nutrient intake (National Geographic, 2011) . The importance of a good diet relies in the energy and nutrients obtained by the consumed food to improve overall health (National Institute of Diabe tes and Digestive and Kindey Diseases, 2019) . The US National Institute of Diabetes and Digestive and Kidney Diseases recommends seeking nutrition resources to help manage different health conditions such as diabetes, obesity, and kidney disease. Unfort unately, not all diets contain the necessary nutrients to maintain healthy dietary (Off ice of Disease Prevention and Health Promotion, 2015) . A combination of these deficits and the excessive consumption of refined grains, processed meats, sodium, sugars, and trans - fat, creates the (Mozaffarian, 2011) . Changes in lifestyle behavior have significantly contributed to the rise of rates of non - communicable diseases, which are also known as chronic diet - related diseases (Office of Disease Prevention and Health Promotion, 2015) . Cardiovascular diseases, diabetes, obesity, and cancer, are some of the examples of chronic disea ses that could be caused by poor diet quality (World Health Organization, 2003) . 71 In fact, the 2015 - 2020 Dietary Guidelines states that 117 million Americans have one or more preventable chronic disease, which many relating to poor qual ity diet. Moreover, overweight and obesity rates are steadily increasing, resulting in extremely high cost health treatments, reaching $245 billion for diabetes costs in 2012 alone (Office of Disease Prevention and Health Promotion, 2015) . The American diet, also known as the Western diet, is a high fat, animal protein and sugar one, with very low levels of dietary fiber, vegetables, and fruits (Chiba et al., 2019; Park et al., 2011; Statovci et al., 2017; Tilg & Moschen, 2015; Uranga et al., 2016) . Therefore, it is crucial to perform accurate and reliable dietary assessments for different types of food items present in the Western diet th at will help elucidate the association between these food items and disease risk (Yin et al., 2017) . Different dietary markers of exposure for many foods have been putatively identified ( Cross et al., 2011; Gibbons et al., 2015; K. A. Guertin et al., 2015; Kristin A Guertin et al., . However, many more food items which are a significant part of Wes tern diet, still need to be analyzed to identify potential biomarkers that will help us recognize and potentially prevent, the development of chronic diseases in the US population. 4.1.1 Western Diet The Western diet is commonly described as one with high fat and refined sugars contents. Additionally, it is characterized by a high intake of red and processed meats, sweets, fried foods, refined grains, and overuse of salt (Drewnowski et al., 2013; Drewnowski & Rehm, 2013; Francis & Stevenson, 2013; Hintze et al., 2012; Myles, 2014) . Western diet m to describe the modern standard American diet. It has been observed in different studies that the 72 components of the Western diet lack of nutritional value, and are micronutrient deficient (Hintze et al., 2012; M yles, 2014) . This intake deficit of micronutrients results in levels below the Recommended Daily Allowances (RDAs) and an excess in consumption of energy - dense and nutrient - poor foods (Hintze, Benninghoff, & Ward, 2012) . Studies have shown that 92% of meals in the US exceeded typical energy requirements for a single eating occasion (Urban et al., 2016) . Restaurant foods, which are popular in the Western diet, tend to have large portion sizes as well as high energy density (McCrory et al., 1999; Urban et al., 2011) , which has been causally associated with highe r energy intake. (Diliberti et al., 2004; Rolls et al., 1999, 2005, 2006) . According to NHANES 2005 - 2006, 68% of the top 25 sources of calories among Americans 2 years and older, were high - energy food items (U.S. Department of Agriculture & U.S. Department of Health and Human Services, 2010) . The existence of all these energy - dense food items has been a result of significant changes that global food systems have undergone, resulting in advanced food processing techniques to increase products availability, a ffordability and marketing of highly processed foods (Floros et al., 2010; B. A. Swinburn et al., 2011; Boyd A Swinburn et al., 2011) . Because of the high demand of food supplies and the dietary patterns of consumption of been created, us ing processing conditions that alter their structure, nutritional content, and taste (van Boekel et al., 2010; van Trijp & Ingenbleek, 2010; Wahlqvist, 2016; Zobel et al., 2016) . - because of the different processing techniques and methods that they undergo as part of their preparation for sale to the public. Processed and ultra - processed foods (which differ in the number of processing techniques applied during their production) are the predominant items in modern societies, hence, the heterogeneity of food processing advocates for new 73 classification tools that can facilitate the assessment of food processing on nutritional quality and health. 4.1.2 Ultra - Processed Foods an d NOVA Classification Ultra - processed food is characterized to be inexpensive, highly processed, rich in calories, but low in some essential micronutrients such as mineral and vitamins. Nearly 60% of the calories consumed by the average Americans come from "ultra - processed" foods (Martínez Steele et al., 2016) . During the past 40 years, an increase of 217 to 491 kcal/day per capita food consumption and self - reported energy intake has been reported (Health, 2011; USDA Economic Research Service, 2014) . Studies have shown that ultra - processed foods comprise 57.9% of energy intake (Martínez Steele et al., 2016) . These meals are mostly consumed away from home and have been proposed as a major contributor to risi ng energy intake (Urban et al., 2016) . The consumption of ultra - processed, calorie - dense, and nutrition - poor food, especially those items from the Western diet, is increasing in developed countries (Juul et al., 2018; Setyowati et al., 2018) . The urban lifestyle with limited time, work overload, and busy schedules influences individuals to buy the most convenient, fast, and cheapest meal options. T herefore, prepared, pre - processed, and ready to eat meals are the preferred ones by the urban population. Nearly every food product present in the Western diet is considered a processed food product. Pr ocessing itself do e s not represent a threat to human h ealth. On the contrary, different processes have been focused to ensure food safety and microbial reduction. However, the formation of harmful compounds derived from different processing parameters requires more attention during the manufactur ing of these food products. 74 Therefore, processing methods play a crucial role in the evaluation of the overall chemical safety of the Western diet. Ultra - processed food s undergo , at least, 4 different proce ss steps promoting the formation of molecules such as COPs. Thus, a new food classification method such as NOVA (not an acronym) classification that considers the processing conditions of each food product is needed to facilitate the study and understanding of these ultra - proces sed foods. (Juul et al., 2018; Martínez Steele et al., 2016; Moubarac et al., 2017) and is recognized by the Food and Agriculture Organization (FAO) and Pan - American Health Organization (PAHO) as a valid tool for nutrition and public health research (C. Monteiro et al., 2016; C. A. M onteiro et al., 2019) . NOVA groups foods according to the nature, extent and purpose of the industrial processing they undergo (Moubarac et al., 2017) . NOVA classifies foods and food products into 4 different groups (Figure 4 - 1). Following - Ultra - processed foods (UPF) is formulations typically (C. Monteiro et al., 2016) . Ingredients found only in ultra - processed products include substances not commonly used in culinary preparations, and additives whose purpose is to imitate sensory qualities of group 1 foods or of culinary preparations of these foods, or to disguise undesi rable sensory qualities of the final product. Fast foods (including small, large, and non - chain restaurants) and Ready to eat (RTE) products are also included in this group. The term fast food has been nack bars and restaurants as a quick . A suitable definition could be 75 the sale of mass - produced UPF n time. RTE are products previously processed and packed for sale which require minimum Figure 4 - 1 : NOVA food classification based on food processing The main concern related with the high consumption of UPF relies on food safety. Even tough industrial food processes are generally designed to improve food quality, nutriti on, and safety, processing can lead to unintentional accumulation of substances such as COPs, which are formed because of thermal treatments, light exposure, storage, and aging of food, and may therefore also negatively impact overall safety. 76 4.1.3 C OPs as B iomarkers A wide range of degradative compounds, such as aldehydes, ketones, and peroxides, are produced because of cholesterol oxidation. These compounds are known as cholesterol oxidation products (COPs ) and have been studied because of their potential relationship with different chronic diseases. Maldonado and co - workers (2018) showed that there is a significant amount of evidence that COPs exert biological and pathological activities in both in vitro and in vivo systems, with potential health concerns fo r humans. As it was previously mentioned in Chapter 1, Lee et al., 2009 have suggested that the apparent association of specific COPs with people more prone to suffer of these chronic diseases could be beneficial, as COPs could be used as biomarkers for t hese diseases (Aldini et al., 2010; Alkazemi et al., 2008; Iuliano et al., 2003) . 7 - Keto has been largely detected and quantified in both model and food systems such as infant formula , suggest ing it as one of the most reliable biomarker of cholesterol oxidation (A. Kilvington, Barnaba, Rajasekaran, Laurens Leimanis, & Medina - Meza, 2021; Rodriguez - Estrada et al., 2014) . However, processing methods such as ultra - high temperature (UHT) pasteurization can also generate sidechain COPs (Pikul et al., 2013) , including the demonstrated cytotoxic 25 - reflect specific processing technologies, as s ome early studies have demonstrated . Therefore, more mechanistic, and quantitative studies for a suitable estimation of COPs in food products, and a harmonization of methodologies for their determination is still needed. 77 4.1.4 Effect of Food Processing on Chol esterol Oxidation Temperature and pressure changes, as well as exposure to light applied during processing, and even packaging and storage techniques, are some factors that promote COP formation. Industrial processing (i.e., cooking, pasteurization, canning, etc.) occurs at variable times and temperatures depending on the type of process and food (Maldonado - Pereira et al., 2018) . Food processing temperatures range between 121°C (canning of meats) and 400°C (spray drying - used in the manufacturing of infant formulas) (Meister et al., n.d.; Verardo et al., 2017) , and even t he US Department of Health and Human Services, suggests that meat and poultry should be cooked at a minimum of 162 °C (FoodSafety, 2017) . Moreover, studies show that several cooking methods considerably increase the formation of COPs. Microwave heating (Herzallah, 2005) , pan roasting, oven grilling (Khan et al., 2015) , oil - frying and other forms of cooking (Nielsen et al., 1996) affect the production of COPs. Until today, t here is no existent database of these compound in the US diet. A COP database of the Western Diet that will allow us to identify potential biomarkers of thermal degradation in UPFs will be reported in this chapter . Eventually, this database will help to de velop a nationwide framework of tools for surveillance, inspection, and traceability of COPs in UPF . It will enable knowledge about processing - formed unintentional compounds which will help to improve or even, avoid processing methods to ensure food safety and therefore, wellbeing of the US population. 78 4.2 MATERIALS AND METHODS A total of 6 3 food items were analyzed in this study. A general overview of the materials used and methodology for the analy sis of UP F is provided below. Details for each sample analysis as well as brand information can be found in the Appendix es C and D . 4.2.1 Materials, Chemicals , and Reagents Methanol was from Sigma - Aldrich (St. Louis, MO). Chloroform was obtained from Omni Solv (Burlington, MA), hexane was purchased from VWR BDH Chemicals (Batavia, IL), 1 - butanol and potassium chloride (KCl) from J. T. Baker (Allentown, PA) and diethyl ether was purchased from Fisher Chemical (Pittsburgh, PA). Sodium sulfate anhydrous (Na 2 SO 4 ), Sodium chloride (NaCl) were also purchased from VWR BDH Chemicals. standards of 7 - hydroxycholesterol (7 - OH), 7 - hydroxycholesterol (7 - OH), 5,6 - epoxycholesterol (5,6 - Epoxy), 5,6 - epoxycholesterol (5,6 - Epoxy), triol, 7 - ketocholesterol (7 - Keto) were purchased from Steraloids (Newport, RI), and purified by using aminopropyl (NH 2 ) cartridges (500 mg/ 3 mL) from Phenomenex (Torrance, CA). 4.2.2 Sample C ollection and P reparation Samples were collected and prepared following the same condition and procedure explained in Maldonado - Pereira et al. 2021 . Forty RTE items and 23 FF meals were selected (FDA, 2018) . Additional information regarding the sam this study is available in Appendix C . Eight of the most popular franchisees in the state of Michigan covering more than 75% of the national market (Dunford et al., 2017; Powell et al., 2019; Rwithley, 2019; Tran et al., 2019) . In addition, f ood items and meals were grouped in subcategories according to the fat source as follow: 79 food (BF). Additional food items that did not fit in any of the previous categories, such as potato - products (potato crisps with and w ithout added flavors, French fries from restaurants and takeaway, frozen potatoes pre - fried and fried, and homemade French fries), pasta, salad dressings, and popcorn (sweet or salty) were grouped as other products (O). Once the UPF arrived at the laborato ry, an excel form was filled out with the following information: (1) (RTE or FF); (5) nutritional declaration (energy, fat, saturated fatty acids, carbohydrates, s ugars, fiber, protein, and salt); (6) portion size; (7) list of ingredients; (8) expiring date; and (9) other relevant information. 4.2.3 Sample P reparation FF meals were purchased from each individual franchise and brought to the laboratory for immediate analysis. RTE meals were purchased from different local supermarket stores, and immediately brought to the laboratory. Storage conditions were followed according to the label instructions (fresh foods were kept in a fridge at 4°C and frozen meals were kept at - 20°C or the temperature indicated in the label). All UPFs were analyzed before the expiration date. When further cooking procedures were required, items e study. All the samples were homogenized using an Ultra - Turrax® (Tekmar TP 18/10S1 Cincinnati, OH) for 3 min at 5000 rpm, split and stored accordingly, depending on the food matrix. 80 4.2.4 Lipid Extraction Lipid fraction was extracted according to Folch and coworkers (Folch et al., 1957) cold extraction method with some modifications depending the food matrix. Specific details for analysis are described in the Supplementary Information - I . 30 grams of sample were minced and homogenized using an Ultratourrax with 200 mL of a chloroform:met hanol solution (1:1, v/v) in a 500 mL glass bottle with screwcap. Sample was mixed for 15 min at 300 rpm. Homogenization was performed using a Ultratourrax for 3 minutes. Bottle was kept in an oven at 60 °C for 20 min before adding 100 mL chloroform. After 2 min of homogenization, the content of the bottle was filtered. The filtrate was mixed thoroughly with a 100 mL of 1 M KCl solution. Samples were left overnight at 4 °C. Then, the lower phase containing lipids was collected and dried with a vacuum evapor ator (Organomation S - EVAP - RB, Berlin MA) at 25 in Hg and 60°C. Total fat content was determined gravimetrically. 4.2.5 Thiobarbituric Acid Reactive Substances (TBARS) The method modified by Miller et al (1994), was used to measure lipid oxidation in all UP food s. Some additional modifications were performed to the protocol. 60 mg of fat were weighed into a 10 mL glass test tube with screw cap and the following reagents were added: 100 L BHT solution (0.2 mg/mL in water) and 4.9 mL extracting solution (10% TCA i n 0.l M H 2 PO 3 ). Each sample was vigorously vortexed in the test tube. Sample blanks were analyzed for each sample. A standard curve was prepared using 0 - 5 mL TEP solution (10 M). Test tubes were incubated in the dark for 15 - 17 hr . at room temperature (25°C). TBARS were expressed as g malondialdehyde (MDA)/g fat sample (dry weight). No adjustment of total volume was needed since fat sample did not contain moisture. 81 4.2.6 Total Cholesterol and Phytosterols Content It is important to ment ion that the FHEL has developed a simultaneous quantification method for both compound groups: Cholesterol and phytosterols (Alice Kilvington, Maldonado Pereira, Torres Palacios, & Medina Meza, 2019) . Cold saponifi cation at room (Bonoli et al., 2008) . Two hundred mg of fat were placed into a glass tube and dried under a nitrogen - - hydroxycholesterol (19 - OH) were added as internal standards. Ten mL of 1 N methanolic KOH were add ed and vortexed. The unsaponifiable fraction was collected and sodium sulfate was added. The mixture was left standing for 2 h and then filtered over a bed of sodium sulfate and collected in a round bottom flask. The solution was then dried in a vacuum eva porator. The sterols were then collected in diethyl ether and dried with a stream of nitrogen. One mL of 4:1 hexane: - hundred microliters (µL) were derivatized by drying with nitrogen and adding 100 µL of pyridine and 100 µL of resuspended in 1 m L hexane. Two µL were injected into a GC coupled to a MS (GC - MS) (Shimadzu GCMS - QP 2010 SE, LabSolutions GCMS Solution Version 4.45). Injector 1 min. Helium was used as a carrier gas with a pressure of 134 kPa. The sterol identification was done by comparing retention times and mass fragmentations from pure standards. Quantification was done by comparing peak areas using the internal standards. 82 4.2.7 C OPs Quantification The remaining 900 µL of the unsaponifiable fat were enriched in the sterol oxidation products by solid - phase extraction (SPE), using NH2 Strata® SPE cartridge. Their purification by NH 2 - SPE was performed according to Rose - Sallin, Huggett, Bosset, Tabacchi & Fay, with slightly modifications. Samples were diluted in 500 L of n - hexane :ethyl acetate (95:5, v/v), followed by the activation of the SPE column by adding 3 mL of n - hexane and 2 - 3 mm of sodium sulfate, and allowed to settle in the column. Enriched samples were dried using a nitrogen stream and then derivatized as described above. One µL of the sample were injected into a GC coupled to a MS (GC - MS) (Shimadzu GCMS - QP 2010 SE, LabSolutions GCMS Solution Version 4.45). The injector temperature compounds was performed using retention times and mass f ragmentations from commercial standards. Using the TIC method, the quantification of the total sterols content was done by comparing the peak areas to the internal standard, 19 - OH. The Selected Ion Monitoring (SIM) method was used to profile the COPs in th e IF. The injector temperature ressure of 49.2 kPa and a column flow Identification of compounds was done using retention times and mass fragmentations from commercial standards. 83 4.3 STATISTICAL ANALYSIS Descript ive statistics were calculated overall and by category. Both mean, and confidence interval (95%) were reported. All the statistical analysis were computed using SPSS v.26 (IBM). 4.4 PRELIMINARY RESULTS A set of UPFs samples together with several homecooked pla tes were tested at the methodology. O ver 266 food items from the Total Dietary Study (TDS) - FDA inventory (Kulig et al., 2016) into: a) High priority, products considered as essential items in every household (i.e., milk, eggs, butter, etc.), b) Ready - to - eat (RTE) food items, representing pre - cooked meals which require simple and fast cooking processes (i.e., microwaving, oven - warming etc.), and c) Fast Foods (FF), representing meals that can be purchased at FF chains. Over 150 homecooked meals (n= 168 , 3 reps each) were prepared following the high priority category. A total of 120 different RTE food items (n= 40 , 3 reps per item) were purchased from 3 of the top retail stores in the US available in the area (i.e., Walmart, Kroger, and Meijer). FF items (n= 22 , 3 reps each) were obtained from the top 10 most consumed FF (O'Callaghan et al., 2014) . Samples were tested for total fat, cholesterol, and eight COPs (referred in this test as )), and phytosterol contents. Secondary oxidation products were analyzed through (TBARS) assay and total SFA, MUFA, and PUFA were quantified. 84 4.4.1 Dietary S L evels The overall dietary cholesterol varied from 48.2 mg/100 g of fat (± 5.9, O8 - FF ) to 1,147.7 mg/100 g of fat (± 160.8, Baby food). S3 - FF contained the highest cholesterol value, followed by MP15 - FF (both from the FF group). In addition, home - cooked bacon (pan - fried) showed the highest cholesterol oxidation percentage with 96% compared to the raw product. Oven - roasted pork ham had a 64% of oxidized cholesterol and boiled frankfurter showed just 15%, which suggests a relationship between the cooking method and the oxidation percentage, involving different factors such as temperature con ditions and the food matrix. Even though cholesterol was the most abundant sterol in these samples, stigmasterol and campesterol were also identified and quantified. O7 - FF and MP14 - FF contained the highest amounts of stigmasterol (224.87 and 209.33 mg/100g fat, respectively). Campesterol quantities were led by O5 - FF and O9 - FF (192.00 and 184.08 mg/100 g fat, respectively; and even quantities of brassicasterol were detected in O9 - FF and MP17 - FF (44.19 and 27.56 mg/100 g fat, respectively). In addition, MP3 - R TE showed the highest stigmasterol content with 259.84 mg/100g fat. Results confirm that sterols oxidation are triggered by light (Maerker & Jones, 1993b; I. G. Medina - Meza & C. Barnaba, 2013; Ilce Gabriela Medina - Meza et al., 2012) , heat (Chien et al., 1998; D. Derewiaka & Molinska nee Sosinska, 2015; Tai, Chen, & Chen, 1999) , radiation (Maerker & Jones, 1993a; L. L. Smith, 199 6) , metal ions (Hur et al., 2007a; Jo et al., 1999) , and other agents that have the ability to lower the energetic requirements for the reaction to occur (Beltran et al., 2004; I. G. Medina - Meza & C. Barnaba, 2013) . 85 4.4.2 Dietary O xidized S terols DO x S As expected, the concentration of DOSs was positively associated with the cholesterol concentration (Figure 4 - 2 ). However , the association was far from perfect (correlation=0.504) indicating that food composition and food processing affects the formation o f DO x S from cholesterol. Among the detected DO x S derived from cholesterol, 7 - OH and 7 - OH were the most abundant ones; however, in some highly processed food items, 7 - keto was present in high concentration. This variability suggests a relationship between DO x S and food matrix. On the other hand, among the detected DO x S derived from phytosterols, - sitosterol, oxidize d Figure 4 - 2 : Cholesterol and oxidized sterols 86 - sitosterol was the dominant phytosterol within RTE items. Our protocol detected more than 30 oxidized metabolites. We isolated and quantified the most abundant DO x S in all food products (7 - OH, 7 - OH, 5,6 - Epoxy, 5,6 - Epoxy, Triol, 7 - Keto, and 24 - methylenecholesterol, and 6 - Keto). Using mixed - effects models we estimated the proportion of variance of each of the DO x S explained by food type, category and food item. 4.4.3 Multivariate A nalysis of DO x S A multivariate analysis of the lipid fingerprinting profiles revealed a clear clustering of compounds and food items the DO x S form a clear cluster of compounds (see red square covering rows/columns 3 to 10 in Figure 4 - 3 ). Figure 4 - 3 : Heatmap of the correlation of lipid profiles across food meals 87 Importantly , our protocol provides simultaneous quantification of both DO x S and phytosterols. For F F , other lipid oxidized derivatives were also detected such as phytosterols oxidation products (POPs). Even though cholesterol was the most abundant sterol in these samples, stigmasterol, campesterol and their oxidized derivatives were also present. 4.5 RESULT S FROM THE COPS ASSESSMENT OF UPFS 4.5.1 Fat C ontent in UPFs Fat content of all 63 UPFs were previously reported in Maldonado - Pereira et al. 2021. Fat was varied between food groups (p < 0.001). Dairy was the food group with the highest fat content, (which includes chicken, beef, turkey, pork, and combinations of these meats) . The majority of the UPFs showed a higher fat content compared to the values reported in either their nutritional lab els or webpages. 4.5.2 TBARS MDA content for FF meals and RTE items are shown in Table 4 - 1 . Results were significantly different between food groups . M eat and poultry, dairy, e derivatives, and seafoo d showed the highest MDA contents . N o presence of MDA in the sample was detected for MP 12 - FF , MP16 - FF, MP17 - FF, MP14 - FF , and O8 - RTE . RTE items with the lowest MDA values were: D4 - RTE , D6 - RTE , D2 - RTE , D7 - RTE , E1 - RTE , D5 - RTE , O4 - RTE , S1 - RTE , BF 9 - RTE , O1 - RTE (0.16, 0.20, 0.36, 0.37, 0.56, 1.04, 1.24, 1.26, 1.48, and 1.88 µg MDA/g fat, respectively). 88 4.5.3 Total Cholesterol Content Cholesterol values for FF s and RTE items are shown in Table 4 - 2 . FF meals had the highest c holesterol content s among all UPFs. Sixteen FF meals out of 2 3 showed a higher nutritional webpages, or other Food Nutrition webpages used for this analysis (Eat this much (DeMenthon, n.d.) and Fast Food Nutrition (Lenhoff, 200 5) ). Between food groups, meat and poultry were the group with the highest cholesterol values. Two items ( O10 - FF and O7 - FF ) showed cholesterol values of 90.13 and 268.72 mg/100 g fat, respectively; even when their cholesterol content in each official FF webpage stablished that these items have 0 mg/100 g fat. On the contrary , no cholesterol was detected for O5 - FF and O9 - FF , which aligns with the cholesterol value expressed in the USDA lesterol was detected for O6 - FF even though USDA food database shows a cholesterol value of 222.99 mg/100 g fat. Nine RTE items out of 40 showed a higher cholesterol content compared to the value 126.63 mg/100 g fat MP2 - RTE , 102.60 18.68 mg/100 g fat E1 - RTE , 276.81 1.83 mg/100 g fat D4 - RTE , 307.81 3.54 mg/100 g fat MP3 - RTE , 300.53 20.51 mg/100 g fat E2 - RTE , 2,135.58 168.32 mg/100 g fat MP6 - RTE , 1,247 227.45 mg/100 g fat BF 1 - RTE , and 313.34 27.54 mg/100 g fat BF11 - RTE . BF9 - RTE showed a cholesterol content of 162.73 95 . 67 mg/100 g fat when the USDA food database reported 0 mg/100 g fat. The other 30 RTE items showed a lower cholesterol content than the one expressed in each nutrition fact label. 89 4.5.4 Phytosterols and Tocopherols Quantification Six p hytosterols (Campesterol, Stigmasterol, Brassicasterol, - Sitosterol, and Fucosterol ) , and - Tocopherol were quantified for FF meals and RTE items (APPENDIX F). FF meals showed the highest phytosterol contents (p < 0.001) , however, the abundance of each phytosterol varied between FF and RTE. - Sitosterol , Campesterol, and Stigmasterol were the le ading phytosterols between FF and RTE. However, - Sitosterol was the phytosterol with the highest quantities within the FF meals (343.35 137.26 mg/100 g fat MP18 - FF , 333.41 45.98 mg/100 g fat - O9 - FF , 331.87 34.11 mg/100 g fat O5 - FF , and 296.67 38.94 mg/100 g fat MP11 - FF ). Stigmasterol was the second lead phytosterol between the FF meals with 224.87 30.79 mg/100 g fat O7 - FF , 209.33 40.88 mg/100 g fat MP14 - FF , and 168.10 24.33 mg/100 g fat MP 23 - FF . Lastly, C ampesterol quantites were: 192.00 21.55 mg/100 g fat O5 - FF , 184.08 25.17 mg/100 g fat O9 - FF , and 122.85 18.26 mg/100 g fat MP11 - FF . For RTE, - Sitosterol was also the phytosterol with the highest quantities within the RTE items (626.48 57.01 mg/100 g fat MP3 - FF , 498.98 129.71 mg/100 g fat BF4 - RTE , 435.62 41.06 mg/100 g fat BF11 - RTE , 421.59 137.27 mg/100 g fat BF7 - RTE , 391.77 93.78 mg/100 g fat BF5 - RTE , 236.18 18.19 mg/100 g fat BF12 - RTE ). Campesterol was the second highest phytosterol between the RTE items with 190.88 21.15 mg/100 g fat for BF7 - RTE , 182.76 53.20 mg/100 g fat for BF4 - RTE , and 136.48 47.29 mg/100 g fat BF5 - RTE . Lastly, S tigmasterol was the 3 rd most abundant phytosterol with values of : 259.84 40.55 mg/100 g fat for MP3 - RTE , 43.37 12.51 mg/100 g fat for D3 - RTE , and 42.06 13.92 mg/100 g fat for E1 - RTE . 90 Traces of Fucosterol were observed in O10 - FF , and no presence of - Tocopherol was observed in FF meals . - Tocopherol was quantified in O2 - RTE (0.019 0.012 mg/100 g fat). Brassicasterol was not detected in any of the RTE items. Food groups that contained ample amounts of phytosterols were pork, egg derivatives, 4.5.5 COPs Quantification Twelve different COPs (7 - OH, 7 - OH , 4 - OH , 5,6 - Epoxy, 5,6 - Epoxy, triol, 7 - Keto, 6 - Keto , 20 - OH, 22 - OH, 24 - OH, and 25 - OH ) were determined in FF meals and RTE items by GC - MS operated in SIM mode (Table 4 - 3 and 4 - 4, respectively). Total COPs values in FF meals ranged between 0. 91 and 39 . 13 mg/100 g fat , and from 0.14 to 63.59 mg/100 g fat between RTE . The dominant COP was 7 - OH , followed by 7 - OH . For 5,6 - Epoxy, MP21 - FF (0.070 0.028 mg/100 g fat) and the MP23 - FF (0.040 0.0019 mg/100 g fat) led the list. Moreover, 5,6 - Epoxy was detected in high amounts in MP21 - FF (0.15 0.049 mg/100 g fat) and O10 - FF (0.15 0.021 mg/100 g fat). MP1 5 - FF was the only meal that contained all COPs resulted from the oxidation of cholesterol at carbon - position 7 (C - 7). These are: 7 - OH, 7 - OH, triol, 7 - Keto. Differences between food groups were significant (p < 0.001), being meat and poultry the leading group. 4 - OH was only detected in FF meals and BF1 - RTE was the only baby food to show content of 5,6 - Epoxy and 5,6 - Epoxy. For side - chain COPs, 25 - OH and 22 - OH were the most abundant ones, reaching values of 3.60 mg /100 g fat in baby food samples. D3 - RTE contained traces of 25 - OH. 91 Table 4 - 1 : Unpublished results of MDA concentration in RTE items and FF meals Ready to Eat Food Category Food Item µg MDA/g fat STD Food Category Food Item µg MDA/g fat STD Dairy D1 - RTE 3.58 ± 1.28 Baby food BF1 - RTE - D2 - RTE 0.36 ± 0.23 BF2 - RTE - D3 - RTE - BF3 - RTE 19.42 ± 7.41 * D4 - RTE - BF4 - RTE - D5 - RTE 1.03 ± 0.052 BF5 - RTE 3.30 ± 0.87 D6 - RTE 0.19 ± 0.017 BF6 - RTE 7.95 ± 5.47 D7 - RTE 0.37 ± 0.27 BF7 - RTE 2.47 ± 0.42 D8 - RTE - BF8 - RTE 2.81 ± 1.43 D9 - RTE - BF9 - RTE 1.48 ± 0.24 D10 - RTE 3.22 ± 2.18 BF10 - RTE 9.36 ± 0.51 D11 - RTE - BF11 - RTE 2.51 ± 1.09 Meat & Poultry MP1 - RTE 29.66 ± 8.43 BF12 - RTE 17.87 ± 11.01 MP2 - RTE 12.67 ± 3.42 BF13 - RTE - MP3 - RTE 7.59 ± 0.23 Eggs & egg derivatives E1 - RTE 0.56 ± 0.042 MP4 - RTE 7.73 ± 5.87 E2 - RTE 1.85 ± 1.19 MP5 - RTE 2.12 ± 1.48 Seafood S1 - RTE 1.26 ± 0.66 MP6 - RTE - Others O1 - RTE 1.88 ± 1.46 MP7 - RTE - O2 - RTE 3.03 ± 0.89 MP8 - RTE 6.76 ± 4.83 O3 - RTE 3.83 ± 0.60 MP9 - RTE 23.83 ± 18.69 O4 - RTE 1.24 ± 0.23 Fast Food Meat & Poultry MP10 - FF 4.15 ± 1.14 Seafood S2 - FF 2.66 ± 1.25 MP11 - FF 1.35 ± 0.79 S3 - FF 0.69 ± 0.38 MP12 - FF - Others O5 - FF 7.27 ± 2.61 MP13 - FF 3.43 ± 1.70 O6 - FF 1.84 ± 1.64 MP14 - FF - O7 - FF 2.11 ± 0.90 MP15 - FF 3.64 ± 2.24 O8 - FF - MP16 - FF - O9 - FF 3.22 ± 1.86 MP17 - FF - O10 - FF 6.20 ± 2.40 MP18 - FF 17.01 ± 6.32 MP19 - FF 2.13 ± 0.47 MP20 - FF 1.20 ± 0.71 MP21 - FF 4.36 ± 1.24 MP22 - FF 3.41 ± 1.95 MP23 - FF 1.53 ± 0.69 MP24 - FF 11.20 7.77 *Only processed food 92 Table 4 - 2 : Unpublished results of total fat and cholesterol contents in RTE items and FF meals Food Group Sample ID Total Fat Content (mg/100 g sample) STD Total Fat Content Reference (mg/100 g sample) Cholesterol (mg/100 g fat) STD Cholesterol Reference (mg/100 g fat) Serving Size Dairy D1 - RTE 17.67 2.45 18.40 20.30 15.85 333.33 19.00 g D2 - RTE 26.92 8.47 33.00 196.33 29.43 333.33 21.00 g D3 - RTE 49.71 2.56 50.00 ND N/A 1 tbsp *D4 - RTE 87.62 1.91 78.57 305.90 41.16 272.73 14.00 g D5 - RTE 10.27 0.38 10.00 165.09 30.33 285.71 30.00 mL D6 - RTE 24.16 6.98 28.00 287.08 20.03 300.00 28 .00g D7 - RTE 42.82 1.68 35.00 238.86 133.50 333.33 28.00 g D8 - RTE 12.78 3.04 23.40 243.02 40.22 357.14 0.50 cup (65.00 g) D9 - RTE 1.60 0.31 3.03 368.25 72.77 0.00 1 container (99.00g) D10 - RTE 2.14 0.54 0.96 282.18 52.16 500.00 414.03 g D11 - RTE 12.56 ± 0.38 0.69 16.25 3.59 N S 1 scoop (9.00 g) Meat & Poultry MP1 - RTE 23.45 5.00 20.00 224.75 15.73 333.33 38.00 g MP2 - RTE 17.84 2.65 26.00 648.56 125.37 250.00 32.00 g MP3 - RTE 0.60 0.071 0.65 327.69 28.27 200.00 240.00 mL MP4 - RTE 3.64 0.018 3.30 157.80 55.79 500.00 1 can (236.59 g) MP5 - RTE 3.53 0.55 5.65 396.25 46.38 500.00 11.00 oz (227.00 g) MP6 - RTE 1.37 0.31 1.76 2,023.90 209.36 750.00 0.5 cup (113.40 g) MP7 - RTE 3.61 0.013 0.61 352.50 166.24 583.33 0.5 cup (240.00 g) MP8 - RTE 2.91 0.53 2.87 62.55 8.88 142.86 1 cup (249.00 g) MP9 - RTE 4.61 0.097 4.28 195.13 101.17 181.82 1 cup (257.00 g) MP10 - FF 33.13 5.12 10.00 370.80 55.74 267.22 95.00 g MP11 - FF 19.52 1.46 20.00 229.99 53.08 222.05 4 pieces (64.00 g) MP12 - FF 23.59 3.40 14.00 362.03 8.90 296.86 119.00 g MP13 - FF 9.83 0.45 11.40 446.38 73.73 464.29 1 taco (102.00 g) 93 Table 4 - 2 Food Group Sample ID Total Fat Content (mg/100 g sample) STD Total Fat Content Reference (mg/100 g sample) Cholesterol (mg/100 g fat) STD Cholesterol Reference (mg/100 g fat) Serving Size Meat & Poultry MP14 - FF 11.06 2.09 7.60 299.73 46.37 230.77 1 quesadilla (170.00 g) MP15 - FF 9.95 0.67 9.90 504.08 7.82 245.31 1 burrito (140.00 g) MP16 - FF 12.48 0.30 12.21 742.86 62.13 820.56 1 piece (75.00 g) MP17 - FF 19.39 2.55 18.60 728.22 42.86 600.00 1 piece (60.00 g) MP18 - FF 6.26 1.21 4.58 333.59 105.66 171.43 5.40 oz (153. 09 g) MP19 - FF 8.19 0.78 10.80 310.55 147.36 1,423.08 5.70 oz (161.69 g) MP20 - FF 14.23 2.82 10.50 526.02 61.70 312.92 1 sandwich (187.00 g) MP21 - FF 7.12 1.18 6.51 450.17 13.91 272.73 1 sandwich (71.28 g) MP22 - FF 10.74 4.18 8.79 335.74 109.38 240.74 1 sandwich (94.61 g) MP23 - FF 12.59 1.35 11.50 280.77 38.10 N / A 1 slice (123.00 g) MP24 - FF 10.96 2.70 11.50 266.45 30.50 200.00 1 slice (79.00 g) Seafood S1 - RTE 3.62 0.25 3.38 43.89 1.81 50.00 18.80 oz (532.97 g) S2 - FF 22.82 1.07 14.08 216.98 26.53 204.39 1 sandwich (131.00 g) S3 - FF 21.70 0.15 13.10 405.14 37.49 555.56 5.00 oz (141.75 g) Eggs and derivatives E1 - RTE 77.68 1.95 75.00 98.44 16.35 90.91 1 tbsp E2 - RTE 14.64 1.02 13.91 274.11 40.94 218.75 0.50 cup (115.00 g) Baby food BF1 - RTE 1.80 0.15 1.41 1,247.23 227.45 1,000.00 71.00 g BF2 - RTE 7.28 0.19 4.40 673.84 98.50 833.33 4.00 oz (113.40 g) BF3 - RTE 2.99 0.06 4.40 58.17 8.50 250.00 4.00 oz (113.40 g) BF4 - RTE 3.25 0.23 4.40 319.43 83.74 500.00 4.00 oz (113.40 g) BF5 - RTE 3.23 0.14 4.40 348.65 82.26 400.00 4.00 oz (113.40 g) BF6 - RTE 2.12 0.60 1.56 123.24 29.61 7.81 128.00 g BF7 - RTE 3.09 0.16 4.40 263.77 76.69 400.00 4.00 oz (113.40 g) BF8 - RTE 6.00 0.14 4.90 683.55 310.63 875.00 71.00 g 94 Table 4 - 2 Food Group Sample ID Total Fat Content (mg/100 g sample) STD Total Fat Content Reference (mg/100 g sample) Cholesterol (mg/100 g fat) STD Cholesterol Reference (mg/100 g fat) Serving Size Baby Foods BF9 - RTE 2.79 0.44 4.40 162.73 95.67 333.33 4.00 oz (113.40 g) BF10 - RTE 4.63 0.48 4.40 251.02 57.95 333.33 4.00 oz (113.40 g) BF11 - RTE 3.89 0.23 4.40 313.34 27.54 285.71 4.00 oz (113.40 g) BF12 - RTE 2.90 0.38 4.40 172.87 17.84 333.33 4.00 oz (113.40 g) BF13 - RTE 1.39 0.25 2.35 327.40 87.35 500.00 85.00 g Others O1 - RTE 19.97 1.85 26.60 ND N/A 2 tbsp (30.00 g) O2 - RTE 45.11 6.32 50.00 51.55 11.01 66.67 2 tbsp (30.00g) O3 - RTE 3.65 1.51 5.00 97.51 32.67 333.33 2.5 oz (70.90 g) O4 - RTE 8.70 5.00 5.00 37.95 8.46 285.71 1 package (58.00 g) O5 - FF 13.92 1.58 20.00 ND 0.00 1 medium serving (117.00 g) O6 - FF 16.27 2.86 16.00 ND 222.99 1 biscuit (76.00 g) O7 - FF 6.29 1.34 6.00 268.72 43.02 0.00 3 hotcakes (149.00 g) O8 - FF 18.16 0.75 17.30 16.42 8.31 5.87 1 biscuit (49.00 g) O9 - FF 15.96 0.21 15.00 ND 0.00 1 order (34.99 g) O10 - FF 1.46 0.52 3.11 90.13 21.47 0.00 1 order (16.85 g) N/A = not apply, ND = not detected , NS = not shown in nutritional labe l, *Only processed food 95 Table 4 - 3 : Unpublished results of COPs concentrations in RTE items Dairy 7 - OH 7 - OH - OH 5,6 - Epoxy 5,6 - Epoxy 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD D1 - RTE 0.15 0.049 0.14 0.080 ND ND ND ND 0.0 ND ND ND ND 1.05 1.21 63.59 25.32 D2 - RTE 0.084 0.034 0.086 0.026 ND ND ND 0.044 0.029 0.051 0.0050 ND ND ND ND ND 0.55 0.13 D3 - RTE ND ND ND ND ND ND ND ND ND ND ND 0.053 0.020 0.14 0.048 * D4 - RTE 0.64 0.29 0.51 0.21 ND 0.044 0.025 0.050 0.019 0.37 0.13 0.18 0.12 0.046 0.036 ND ND ND ND 2.00 0.92 D5 - RTE 0.19 0.0061 0.12 0.029 ND ND ND 0.11 0.016 ND ND ND ND ND ND 1.29 0.39 D6 - RTE 0.32 0.10 0.27 0.036 ND ND ND 0.11 0.025 0.051 0.0056 ND ND ND ND ND 1.72 0.26 D7 - RTE 0.51 0.26 0.39 0.20 ND 0.023 0.0046 0.058 0.011 0.20 0.094 0.14 0.032 0.046 0.020 ND ND ND ND 1.41 0.52 D8 - RTE 0.039 0.019 0.043 0.018 ND ND ND ND ND ND ND ND ND ND 0.21 0.081 D9 - RTE 0.36 0.056 0.35 0.072 ND ND ND 0.16 0.020 0.10 0.013 0.065 0.038 ND ND ND ND 4.06 0.98 D10 - RTE 2.75 1.88 4.72 3.26 ND 0.20 0.16 ND 1.40 0.92 2.49 1.84 0.19 0.038 ND 0.15 0.068 ND 0.43 0.26 16.09 8.01 D11 - RTE 0.075 0.0094 0.09 0.033 ND ND ND ND ND ND 0.055 0.032 ND ND ND 0.90 0.061 96 Table 4 - 3 Meat & Poultry 7 - OH 7 - OH - OH 5,6 - Epoxy 5,6 - Epoxy 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD MP1 - RTE 5.95 5.43 4.83 4.30 ND 0.10 0.064 0.11 0. 1.93 1.57 0.15 0.076 0.065 0.029 ND 0.068 0.042 ND 0.39 0.32 15.27 12.80 MP2 - RTE 4.20 1.20 3.76 1.06 ND 0.12 0.026 0.13 0.040 1.14 0.41 0.058 0.0071 0.052 0.020 ND ND ND 0.36 0.12 10.25 2.80 MP3 - RTE 0.081 0.019 0.060 0.027 ND ND ND 0.015 0.0084 ND ND ND ND ND ND 0.25 0.027 MP4 - RTE 0.96 0.14 1.13 0.19 ND ND ND 0.29 0.021 0.22 0.072 ND ND 0.10 0.034 ND 0.14 0.095 6.99 1.85 MP5 - RTE 0.42 0.11 0.32 0.024 ND ND ND 0.16 0.099 0.082 0.012 0.079 0.020 ND 0.17 0.0081 0.050 0.031 0.11 0.021 2.17 0.22 MP6 - RTE 15.85 3.91 16.07 4.20 ND 0.26 0.12 0.41 0.13 3.75 0.68 2.33 0.37 ND ND ND 0.40 0.084 ND 43.12 8.98 MP7 - RTE 0.14 0.029 0.15 0.060 ND ND ND 0.066 0.044 ND ND ND ND ND ND 0.36 0.13 MP8 - RTE 0.54 0.062 0.47 0.066 ND ND 0.13 0.022 0.12 0.039 0.053 0.020 ND ND 0.66 0.043 ND 0.19 0.0078 3.09 0.16 MP9 - RTE 0.27 0.067 0.20 0.070 ND ND ND 0.085 0.025 ND ND ND ND ND ND 1.36 0.38 Seafood 7 - OH 7 - OH - OH 5,6 - Epoxy 5,6 - Epoxy 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD S1 - RTE 0.052 0.020 0.060 0.013 ND ND ND ND ND ND ND 0.090 0.013 ND ND 0.25 0.026 97 Table 4 - 3 7 - OH 7 - OH - OH 5,6 - Epoxy 5,6 - Epoxy 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD E1 - RTE 0.11 0.051 0.13 0.024 ND ND ND 0.062 0.021 ND ND 0.025 0.017 0.069 0.077 ND 0.22 0.036 0.91 0.15 E2 - RTE 0.47 0.27 0.32 0.19 ND ND ND 0.18 0.088 ND ND ND ND ND ND 1.35 0.60 Baby Food 7 - OH 7 - OH - OH 5,6 - Epoxy 5,6 - Epoxy 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD BF1 - RTE 8.97 5.42 24.61 15.21 ND 0.69 0.36 1.39 0.81 16.69 5.45 2.63 0.46 ND ND ND ND 3.60 1.22 62.55 25.55 BF2 - RTE 2.92 0.73 2.27 0.64 ND 0.044 0.012 0.033 0.0062 0.84 0.34 0.22 0.031 0.11 0.064 ND ND ND 0.24 0.055 8.48 2.46 BF3 - RTE 0.10 0.031 0.065 0.037 ND ND ND ND ND ND 0.65 0.17 0.33 0.11 0.18 0.47 ND 2.65 0.46 BF4 - RTE 1.80 0.22 1.61 0.24 ND ND ND 0.57 0.065 0.27 0.086 ND ND 0.20 0.044 ND 0.16 0.056 7.32 1.08 BF5 - RTE 2.10 0.50 1.94 0.37 ND 0.052 0.021 0.045 0.027 0.59 0.18 0.31 0.091 ND ND 0.10 0.034 ND 0.27 0.16 6.44 1.23 BF6 - RTE 3.73 1.98 0.66 0.12 ND ND ND 0.67 0.42 0.20 0.034 ND ND 0.077 0.031 ND ND 8.04 3.16 BF7 - RTE 2.12 0.16 2.38 0.15 ND ND ND 0.48 0.15 0.39 0.056 ND ND 0.17 0.069 ND ND 6.13 0.30 BF8 - RTE 5.85 1.82 4.91 1.12 ND 0.030 0.019 0.073 0.039 1.68 0.61 0.27 0.10 0.12 0.018 ND ND ND 0.67 0.16 14.02 3.97 BF9 - RTE 0.68 0.12 0.59 0.17 ND ND ND 0.24 0.075 0.21 0.050 ND ND ND ND ND 8.14 5.70 98 Table 4 - 3 BF10 - RTE 2.08 0.63 1.62 0.46 ND 0.027 0.016 0.042 0.032 0.65 0.087 0.35 0.041 ND ND 0.067 0.015 ND 0.27 0.047 6.36 1.36 BF11 - RTE 0.66 0.092 0.58 0.020 ND ND 1.05 0.12 ND 0.12 0.036 ND ND 2.48 1.45 ND ND 5.28 1.49 BF12 - RTE 0.41 0.10 0.40 0.078 ND ND ND 0.18 0.013 0.10 0.044 ND ND 0.18 0.11 ND ND 2.05 0.31 BF13 - RTE 4.22 3.77 1.27 0.89 ND 0.062 0.023 0.10 0.025 1.31 0.83 0.15 0.03 ND ND 0.12 0.017 0.14 0.029 0.47 0.32 11.58 7.07 Others 7 - OH 7 - OH - OH 5,6 - Epoxy 5,6 - Epoxy 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD O1 - RTE 0.12 0.018 0.094 0.023 ND ND ND ND 0.053 0.014 0.023 0.011 ND 0.099 0.027 ND ND 1.56 0.28 O2 - RTE 0.11 0.037 0.089 0.015 ND ND ND 0.035 0.0079 0.060 0.016 ND 0.031 0.020 ND ND ND 0.61 0.12 O3 - RTE 0.57 0.11 0.60 0.14 ND 0.10 0.030 0.055 0.043 0.20 0.060 0.13 0.055 ND ND 0.083 0.0022 ND ND 5.60 1.28 O4 - RTE 1.19 0.78 1.22 0.79 ND 0.066 0.028 0.054 0.053 0.30 0.095 0.17 0.063 0.078 0.020 ND 0.051 0.027 ND 0.15 0.10 9.58 2.73 *Only processed food 99 Table 4 - 4 : Unpublished results of COPs concentrations in FF meals Meat & Poultry 7 - OH 7 - OH - OH 5,6 - E 5,6 - E 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD MP10 - FF 3.06 1.37 2.28 1.21 ND 0.051 0.0052 0.068 0.024 0.92 0.48 0.12 0.057 ND ND ND 0.14 0.094 0.21 0.12 8.64 3.04 MP11 - FF 0.31 0.031 0.29 0.048 ND ND ND 0.12 0.052 0.033 0.015 0.045 0.034 0.11 0.060 0.21 0.17 ND 0.17 0.013 1.91 0.084 MP12 - FF 1.00 0.29 0.72 0.28 ND ND ND 0.35 0.083 0.030 0.020 0.050 0.0052 ND ND 0.10 0.024 0.11 0.044 3.18 0.65 MP13 - FF 0.26 0.023 0.28 0.041 ND ND ND 0.077 0.16 0.024 0.012 ND ND ND ND 0.12 0.036 1.67 0.20 MP14 - FF 0.75 0.044 0.55 0.086 ND 0.056 0.038 0.13 0.041 0.54 0.21 0.21 0.037 0.070 0.021 0.13 0.036 0.20 0.030 ND 0.2 2 0.038 14.64 11.61 MP15 - FF 1.89 0.66 0.78 0.10 0.49 0.030 0.061 0.013 0.11 0.016 0.86 0.33 0.12 0.040 ND ND ND ND ND 5.02 1.08 MP16 - FF 0.69 0.20 0.75 0.22 ND 0.056 0.037 0.044 0.023 0.20 0.033 0.053 0.017 ND 0.050 0.017 ND ND ND 3.90 1.00 MP17 - FF 0.19 0.44 0.19 0.037 ND ND ND 0.63 0.018 0.031 0.018 ND ND ND ND ND 1.55 0.38 MP18 - FF 3.04 0.32 0.99 0.42 ND 0.023 0.011 0.029 0.0099 0.57 0.089 0.067 0.023 0.039 0.014 ND ND ND ND 5.72 0.99 MP19 - FF 0.48 0.044 0.34 0.036 ND 0.030 0.020 0.028 0.0057 0.20 0.040 0.11 0.026 0.021 0.0069 ND 0.10 0.032 ND 0.049 0.029 1.75 0.077 MP20 - FF 0.18 0.060 0.16 0.040 ND ND ND 0.057 0.035 0.037 0.031 ND ND ND ND 0.090 0.055 0.91 0.11 MP21 - FF 4.01 0.081 3.50 0.14 ND 0.060 0.019 0.12 0.041 1.42 0.24 0.11 0.038 0.069 0.052 0.019 0.0073 0.019 0.016 0.18 0.029 0.12 0.009 9 10.40 0.71 MP22 - FF 1.81 0.23 2.61 0.29 ND ND ND 0.71 0.034 0.071 0.019 ND ND ND ND ND 5.40 0.56 100 Table 4 - 4 MP23 - FF 16.80 9.61 10.23 4.14 0.41 0.22 0.21 0.078 0.52 0.28 ND 0.60 0.32 0.22 0.081 0.16 0.045 0.43 0.11 ND 1.29 0.64 39.13 18.94 MP24 - FF 0.14 0.036 0.18 0.035 ND ND ND ND 0.036 0.012 ND ND ND ND 0.15 0.064 2.53 0.33 Seafood 7 - OH 7 - OH - OH 5,6 - Epoxy 5,6 - Epoxy 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD S2 - FF 0.33 0.075 0.23 0.029 ND ND ND 0.15 0.012 0.092 0.029 ND ND ND ND 0.16 0.079 2.66 0.44 S3 - FF 1.37 0.83 1.27 0.72 ND 0.055 0.040 0.18 0.037 0.43 0.25 0.12 0.069 0.081 0.034 0.12 0.065 0.20 0.025 ND 0.17 0.042 7.27 2.85 Others 7 - OH 7 - OH - OH 5,6 - Epoxy 5,6 - Epoxy 7 - Keto Triol 6 - Keto - OH 22 - OH 24 - OH 25 - OH Total COPs (mg/100 g fat) STD O5 - FF 0.096 0.036 0.093 0.033 0.031 0.0090 0.021 0.0041 ND 0.084 0.028 ND ND ND ND 0.15 0.039 ND 3.16 1.11 O6 - FF 0.52 0.31 0.30 0.13 ND ND ND 0.26 0.18 0.10 0.017 ND 0.16 0.10 0.18 0.017 ND tr 1.97 0.61 O7 - FF 0.81 0.038 0.60 0.044 ND 0.068 0.036 0.080 0.018 0.22 0.021 0.075 0.015 ND 0.11 0.042 ND ND ND 2.40 0.14 O8 - FF 0.11 0.073 0.096 0.045 ND ND ND ND 0.021 0.013 ND ND 0.024 0.019 ND 0.16 0.033 3.14 1.56 O9 - FF 0.099 0.037 0.082 0.033 ND ND ND 0.056 0.027 0.033 0.0065 ND 0.052 0.013 ND ND 0.19 0.049 9.62 2.24 O10 - FF 0.42 0.052 0.37 0.060 ND ND 0.10 0.022 ND 0.041 0.017 ND ND 0.52 0.030 ND 0.15 0.011 2.33 0.21 101 4.6 DISCUSSION 4.6.1 Fat in UP Fs From the 1 9 RTE items that showed higher amounts of fat compared to their nutritional fact label or the FDA database, 10 are either dairy products ( Figure 3 - 1) or products that contain a dairy product as an ingredient ( D4 - RTE , S1 - RTE , D5 - RTE , D7 - RTE , O4 - RTE , MP7 - RTE , D10 - RTE , D11 - RTE, MP8 - RTE , MP9 - RTE ). All these RTE items are prepared with milk, which could contain up to 3.25% of fat (whole milk), in addition to other ingredients with high fat content such as oils. Another 5 of the RTE items with a high fat content are baby foods: BF2 - RTE , BF8 - RTE , BF 6 - RTE , and BF10 - RTE . Some of the ingredients of BF10 - RTE are: canola oil and ground chicken, which could contribute to its high fat content. However, this means that children are also exposed to develop differen t chronic disease, such as obesity (Ogden et al., 2014) , due to the unmonitored con sumption of these foods manufactured for infants and children which contains such high levels of fat. In fact, the US Centers for Disease Control and - quarter of 2 - to 5 - year - old children in the United States h ave overweight or obesity defined as at or above the 85th and 95 th percentiles, respectively, for age - and sex - (Emond et al., 2020) Three additional RTE items with high fat content contained meat (specifically, beef) as their principal ingredient. Beef is a meat high in fat content that can be observed from roasted rib roast large end which contains 24 g per serving size (3 oz) (USDA, 2011) , to ground beef which can contain a maximum 30% fat allowed by the USDA (USDA, 2016) . E1 - RTE was the last RTE item with a high fat content. Mayonnaise contains egg yolk which is the 102 part of the egg with a higher fat content. One small egg could have 5 g per serving size (1 egg = 50g). (ENC, n.d.) From the FF meals, the obtained fat content was drastically different from the value obtained from the USDA food datab meals showed a fat content higher than the one mentioned in either the USDA food to be served in large portions, conta ining high levels of saturated fat and added sugars. (Harris et al., 2013; Rosenheck, 2008) Most of these FF meals are prepared with several different ingredients su ch as oils, eggs, high - fat meats; all of them containing high fat content themselves, resulting in a meal with an overall high fat content. RTE and FF meals are the main contributors of fat accumulation in the body due to their high fat levels. (Mohiuddin & Nasirullah, 2020 ) Fat accumulation in the body is directly related to different chronic diseases such as obesity, diabetes, hypertension, atherosclerosis, among others (Shori et al., 2017) . Therefore, the quantification of total fat in RTE items and FF meals is of great importance to corroborate the actual fat intake of adults and children who con sume UP foods in the USA. 4.6.2 Secondary Oxidation Products (TBARS) TBARS assay has been widely used as a validation technique for lipid oxidation in food (Miller et al., 1994; Permal et al., 2020; Zeb & Ullah, 2016; Y. Zhang et al., 2019) . The determination of secondary oxidation products by MDA quantification confirms the extent of lipid oxidation in a food sample. No differences were observed between FF and RTE samples but were observed b etween food groups (p < 0.0 0 1). However, since this test was mainly developed for comparison between solely raw and cooked meat samples (not fat samples of 103 entire meals with a mix of different ingredients as it was in our case for FF meals and RTE items), using an entire piece of the food matrix, MDA was not able to be quantified in some of our samples. Our hypothesis is that after a certain amount of time and due to all the temperature changes, that these specific meals undergo before, during, and after th e ir final cooking step at home, all the unstable MDA molecules tend to convert in other molecules such as aldehydes and ketones, hinderin g its measurement during the TBARS assay. In general, MDA could not be detected in more FF meals than RTE items. Eight RTE items were not tested for TBARS. 4.6.3 Total Cholesterol FF was the category with the highest cholesterol content (p < 0.01). Overall, cholesterol values were d ifferen t between their reference values stablished in their nutritional fact label or the FF meals webpages. Sixteen out of 23 FF meals showed differences up to 179.78% in cholesterol content. From these 16 FF meals with high cholesterol content, 2 of these meals ( O10 - FF and O7 - FF ) showed chole sterol quantities of 90.13 and 268.72 mg/100 g fat, respectively, even though their values from the data base O9 - FF and O5 - FF showed 0 mg/100g fat of cholesterol which was confirmed by the wheat flour which contains no cholesterol. MP23 - FF had a total cholesterol content of 504.08 mg/100g fat; however, its value could not be confirmed by any source since this item has been dis continued from the market. As it was mentioned above, FF meals also showed for most of their items, a higher fat content compared to the values obtained from the USDA food database, or each individual 104 FF webpage or Eat this much and Fast - Food Nutrition we bpages. A positive relationship can be seen with fat content and cholesterol content, which could help us to further understand COP s formation in foods. The food group with the highest average cholesterol content was meat and poultry, reaching a value of 440.69 mg/100 g fat. The 2 nd group was baby foods with cholesterol contents up to 380.40 mg/100 g fat (Figure 4 - 4) . Figure 4 - 4 : Distribution of cholesterol between food groups 4.6.4 Phytosterols and Tocopherols in UPFs The food matrix and ingredients added to the food item throughout its preparation plays an important role in this phytosterols distribution for being compounds mostly derived from 105 oils and vegetable origin ingredients. Canola oil, olive oil, avocado, and o ther vegetables are some examples of ingredients that are added to different RTE items and FF meals to Overall, FF contained higher amounts of phytosterols . Food groups with the h ighest phytosterols content were: baby foods , and T he RTE item with the highest content was MP3 - RTE. Figure 4 - 5 : Distribution of phytosterols between food groups These items, especially baby foods, contain high amounts of canola and other vegetable derivatives which are rich in phytosterols, hence, it was expected to find several phytosterol in these samples; specifically , - Sitosterol. Sixteen of the RTE items did not presented any phyt osterol content which are items were meat, cheese and other animal - origin ingredients 106 - Sitosterol. A total of 21 FF meals showed a total phytosterol amount of 36.45 17.21 mg/100g fat. O9 - FF and O5 - FF were the 2 FF meals with the highest total phytosterols content (561.67 83.62 and 523.88 55.47 mg/100g fat, respectively), followed by MP11 - FF with 419.52 56.86 mg/100g fat. French fries and chicken nuggets are cooked in the industry using vegetable oil; hence, it was expected to find several phytosterol in these samples; specifically, - Sitosterol. Just 2 FF meals did not show any phytosterol content ( MP10 - FF and M P12 - FF ) mainly because of its dominant meat composition which in turns results in a high cholesterol content as it was previously described. No FF meal showed presence of Fucosterol or - Tocopherol. The presence of phytosterols in FF meals and RTE is of great importance since these compounds are a lso known to be susceptible to different processing condition such as temperature changes, light exposure, and other factors that could result in the formation of other type oxidation products, similar to COP s. ( Kilvington et al., 2019; Maldonado - Pereira et al., 2018) 4.6.5 COP s Occurrence in the Western Diet UP Fs foods are produced under different and variable conditions. M ost of the time , t hese food products undergo several process ing steps as part of the desired final product. Hence, formation of COP s is known to be promoted as an effect of these constant parameters variability during UP manufacturing. (Guardiola et al., 2002; Maldonado - Pereira et al., 2018) Therefore, the database created in this chapter is vital to know their occurrence in the 107 Western d processing techniques affect and promote their formation. Differences in food composition and processing conditions can be observed in both FF meals and RTE items. In FF meals, total C OP s content reached values up to 39.13 mg/100g fat ( MP2 3 - FF ). On the contrary, the lowest amount was 0. 91 mg/100g fat ( MP2 0 - FF ). For RTE items, the highest value for total C O P s was 63.59 mg/100 g fat for D1 - RTE , followed by BF1 - RTE with 62.55 mg/100 g fat. The lowest C O P s contents were: 0 .14 mg/ 100 g fat for D3 - RTE . A total of 12 individual C O P s were found in our samples: 7 - OH, 7 - OH, - OH 5,6 - Epoxy, 5,6 - Epoxy, Triol, 7 - Keto, 6 - Keto - OH, 22 - OH, 24 - OH, and 25 - OH . The food group with the highest amounts of COPs was meat and poultry (Figure 4 - 5). Figure 4 - 6 : Distribution of COPs between food groups 108 Moreover , MP1 5 - FF contained all COPs resulted from the oxidation of cholesterol at carbon - position 7 (C - 7). These are: 7 - OH, 7 - OH, triol, 7 - Keto. As it was previously mentioned, a relationship with C O P s formation and food type is significant (p < 0.001) and could help us to better understand COPs formation p athway in all these food items. These thermal changes vary among the food matrix, and the cooking methods and conditions . H ence, COPs formation, which are the predominant group within the lipid oxidation products, becomes not only directly related to the f ood matrix composition, but also to the processing method, packing and storage conditions, which are standardized and completely different from one franchise or brand to another. For RTE items, a total of 11 COPs were found in our samples: 7 - OH, 7 - OH, 5, 6 - Epoxy, 5,6 - Epoxy, Triol, 7 - Keto, 6 - ketocholestanol, - OH, 22 - OH, 24 - OH, and 25 - OH . No presence of - OH was found. This difference marks a distinction between RTE items and FF meals. However, since this is the first study performed to investigate FF meals and RTE items and there are not many studies focused on the potential relationship between COPs , and FF meals and RTE items, there is not much informa tion that we could use to compare our results and deeply evaluate all these changes observed after our analyses. For general items such as egg products, French fries and beef hamburger, some studies can be found ( (Bonoli et al., 2008; Broncano et al., 2009; Brzeska et al., 2016; Hur et al., 2007; Ma ldonado - Pereira et al., 2018; Medina - Meza, Rodriguez - Estrada, et al., 2014; Medina - Meza & Barnaba, 2013; Nielsen et al., 1996; Paniangvait et al., 1995; Won Park & Addis, 1987; Zardetto et al., 2014; W. B. Zhang et al., n.d.) and used for general compar ison. It is known that the presence of 7 - Keto in oxidized samples marks the completion of the lipid oxidation mechanism, being 7 - Keto one of the last COPs formed based on reaction 109 rates previously studied (I. Medina - Meza & C. Barnaba, 2013) . For most of our samples, 7 - Keto was found in considerable quantities. At this point, a kinetics analysis to validat e these reaction rate values is required to obtain this critical information regarding COPs formation and development in FF meals and RTE items. 4.7 CONCLUSION S Fat was significantly higher (p < 0.001) in FF meals compared to RTE items. Also, fat was positive ly correlated to the amount of SFAs (p < 0.001), while PUFAs are positively correlated with phytosterols content (p < 0.001), which at the same time, were directly related with the use of vegetables oils and other ingredient such a fish, avocado, among oth ers, which contain high PUFA levels. High amounts of fat tend to result in a high amount of cholesterol for those FF meals and RTE items dominated by animal - origin ingredients. However, several items that had a reference cholesterol value of 0 mg/100 g fat, showed actual values other than 0, meaning there is some in congruency between the information general, higher than those of FF meals. However, the inability of detecting MDA in FF meal could mean that the lipid oxidation extent was higher in the FF meals, because of the possible presence of other secondary oxidation products formed after the decomposition of MDA molecules such as aldehydes, ketones, among others. Lastly, cholesterol was positively related with COPs , but some var iability between food matrix es and cooking parameters was observed . The UPF with the highest COPs amount was D1 - RTE . However, the food group with the highest tot a l COPs value was the baby food group., 7 - OH and 7 - OH were present in all FF meals, except for D3 - RTE. A ll baby foods showed presence of 7 - OH, but phytosterols basically dominated this group of RTE items. 110 CHAPTER 5 : LIPID OXIDATION SP E C I ES IN THE WESTERN DIET A HEALTH AND NUTRITION THREAT 5.1 OVERALL SIGNIFICANCE This study directly impacts four main areas: Food safety, Nutritional value, Food Market, and Public Health described in detail in the following sections and Figure 5.1. This project enhances the current nutritional knowledge of oxidized lipid on food and food products provid ed to the consumers through the nutritional label, by generating information of the presence of C O P s as well as their relationship with chronic diseases . It is expected that the decision - making process of laws and regulations ensuring food safety through t he implementation of a standardized and correct procedure of processing techniques will be improved, considering these potentially hazardous compounds. Figure 5 - 1 : Study's significance in the three main areas: Public Health, Nutrition, and Food Market 111 5.2 LIPID OXIDATION AND FOOD SAFETY For decades, food safety has been focused on avoiding food borne illnesses and microbial reduction (FoodSafety.gov, 2020a) . Therefore, different processing conditions and methods have been developed following the suggestions and conditions required to reduce the risk of getting any food - sick or microbial infection. Unfortunately, the food market has never considered other potentially harmful compounds that could result from current processing techniques in the food industry. They focus on complying with the regulations stablished by the government without knowing the existence of other hazardous species. This is the case of COPs and other lipid oxidation species that have been overlooked for the past 30 years despite their associa tion with different chronic diseases (Bang et al., 2008; S. Lemaire et al., 1998; Lemaire - Ewing et al., 2005; Maldonado - Per eira et al., 2018; Snelson et al., 2021) . Chapter 1 through 4 were developed to pr point of view of food safety by their adverse health effects which have been reported recently in several studies (Joshi, Darshane, & Beke, 2020; Snelson et al., 2021) . Thes e chapters suggest a deeper analysis of the unpredictable formation of these lipid oxidation species in food because of the variability in food processing technologies and cooking conditions to ensure a chemically safe food product. 5.3 LIPID OXIDATION AND NUTRITIONAL V ALUE T he effect of processing conditions in the food quality was demonstrated in Chapter 3. Chapter 3 highlights the nutritional changes in fatty acids composition resulting from the variability of parameters as part of man ufacturing processes used in the food industry. This chapter proved that these conditions, which are developed to ensure the manufacturing of a safe and secure food product, play a crucial role in the overall nutritional content of foods. A 112 deeper analysis of the nutritive changes and the unpredictable formation of these lipid oxidation species in food because of the variability in food processing methods and conditions was suggested to ensure a nutritious product. This project enhanced the current nutritio nal knowledge of food and food products provided to the consumers through the nutritional label, by generating information of the presence of COPs and other antioxidant molecules. Dietitians and nutritionist will benefit from this study by obtaining a deta iled chemical and nutritional report of the most consumed 63 ultra - processed foods in the US which will help them to better understand and acknowledge the presence of these potential hazardous species in the US diet. It is expected that this information wi ll start to be considered in the evaluation of healthy diet options for the US population. 5.4 LIPID OXIDATION AND THE FOOD LAW F ood law governs food production, distribution, and consumption t o protect the consumers against unsafe, adulterated, and misbranded food (Kotwal, 2016) . It is also an important area impacted by this study. The most consumed UPFs by the US population are RTE and FF, which account for 60% of the calorie intake per day (C. a. C. G. a. L. M. a. L. M. L. a. M. P. Monteiro, 2019) and are considered one of the most cheap and available food options. Therefore, the food industry and policy makers will benefit from this novel information about the COPs content in UPFs according to different food technologies. New information regarding the processing and manufacturing of their products will help to start Policy makers will obtain new and critical nutritional, health, and even economical knowledge regarding food safet y. This study observed an inverse trend between prices and calories per serving which emphasizes the effect of product affordability and food security 113 with dietary patterns. Laws and regulations can be modified considering new health threats such as the ad verse effects of these lipid oxidation species such as COPs. In general, the food industry, including law and policy makers will acquire information that will improve the decision - making process of new food laws and regulations to ensure the well - being of us citizens. 5.5 LIPID OXIDATION SPECIES AND HUMAN HEALTH The significance of this study relies mainly on COPs and other lipid oxidation species such as POPs (specifically, their effects related to infant formula consumed by infants during their first months o f life) as a new Public Health issue. Chapters 1 and 2 remarked the adverse effects on human health of COPs and highlighted the need of a database of these compounds in the Western diet, which was addressed in Chapter 4. This project increased the current knowledge of the occurrence, and prevalence, of COP s in the Western diet , by the creation of a complete database of COP s present in the Western diet available to be used in new toxicology studies, by food scientists, food engineers, nutritioni sts, dietitians, as well as the general public. The new information derived from this project, will provide a baseline for future nutritional, toxicological, and epidemiological studies in COP s that will expand current medical knowledge of chronic diseases related to these compounds. Novel nutritional quality information of food products , the presence of C O P s and their relationship with chronic diseases can be available to consumers, with the possibility to change/modify their eating patterns to healthier a lternatives . The use of this database could create a paradigm shift regarding the use of calorie content as major parameter when designing diets , allowing for more individual and specific nutritional and medical treatments. Additionally, Chapter 4 suggeste d an Absorption, Distribution, Metabolism and Excretion 114 (ADME) study as the next specific step to address COPs as a potential health threat. Results from the ADME study will enable the performance of more detailed exposure assessments, and eventually, risk assessments. 5.5.1 Dietary Exposure Assessments as a Public Health Tool An exposure model is an e valuating method of the intake of a compound of concern over a specified period. It d escribes variability and uncertainty across populations and sub - populations considering different socio - economics parameters such as age, gender, education level, annual income, race, ethnicity, etc. It is also p art of the risk assessment response mode estimate risk . Exposure can be expressed as a function of magnitude, duration, and frequency. The significance of performing an exposure assessment is described in (Figure 5.2). Figure 5 - 2 : Importance of an Exposure Assessment 115 5.5.2 SHEDS - HT Exposure Model The Stochastic Human Exposure and Dose Simulation (SHEDS) was the exposure model modified and tested using COPs values obtained from this study. SHEDS was developed by the US Environmental Protection Agency (EPA) and has been used to create different versions of predictive models for a variety of chemicals. It can characterize in detail the variability and uncertainty in population exposures using demographic, exposure factor, and chemical a pplication data, in combination with human activity and location information . SHEDS has been successfully applied for consumer product ingredients and agricultural pesticides such as organophosphate and pyrethroid pesticides (Buck, Ozkaynak, Xue, Zartarian, & Hammerstrom, 2001; Hore et al., 2006; Tulve et al., 2011; V. Zartarian et al., 2012; V. G. Zartarian et al., 2000) , arsenic (J. Xue et al., 2006; J. P. Xue, Zartarian, Wang, Liu, & Georgopoulos, 2010; V. Zartarian et al., 2006) , and methyl mercury (J. P. Xue, Zartarian, Liu, & Geller, 2012) . However, it has not been utilized yet for any dietary exposure assessment. SHEDS - High Throughput (SHEDS - HT) was developed as a leaner and more versatile vers ion that included the direct ingestion pathway and allows it to be applied quickly to a large number of chemicals. SHEDS - HT was chosen because of its benefits and characteristic s : It is run in R (mostly available for free) It has p otential to generate population distributions of daily level exposures and intake doses (mg/kg - body weight/day) for a range of chemicals present in residential environments, foods, and drinking water in a HT capacity. 116 It models a ctive chemical exposure pa thways for a given compound for th e simulated population Calculates aggregate chemical exposures for each individual Uses Monte Carlo methods to assign relevant exposure factors and cohort - matched activity and food intake diaries to each person Ingestion exposures are based on a fraction of mass that is ingested during the consumption of the food item. Figure 5.3 shows the three main input categories that are required to run SHEDS - HT: (1) Popula tion Characterization : US Census, and Dietary diaries from NHANES - WWEIA: 1999 - 2006 , (2) Chemical Data : c hemical and physical properties of tested COPs , and their Figure 5 - 3: SHEDS Input Categories (Isaacs et al., 2014) 117 concentration in the foods tested in this study; (3) Consumer Product Data : Since this is a Dietary Exposure Assessment, this category was not needed for our exposure assessment. 5.5.2.1 How D oes SHEDS W ork? The model includes 4 main files that were optimized to include the UPFs, COPs concentrations and their chemical and physical properties needed to en able their analysis: 1. chem_props.txt = contains all the chemical and physical properties of the compounds analyzed . 2. diet_diaries.txt = contains daily intakes in [g/day] for various food s . Each record corresponds to one person for one day. The default diary file contains aggregated summaries of food consumption data from the NHANES - What We Eat In America Study (1999 - 2006, USDA 2014; NHANES - WWEIA). Food consumption in grams is given for each of the SHEDS - HT default foods, for each person in the study. The variables on the file are the age, gender, body weight in [kg], and the intake quantities. The food groups are indicated by short labels (usually two characters). These are matched in SHEDS with distributions from the s ource.chem ical s ourc e.chemicals file. The user may use their own choice of food groups provided both input files are consistent . 3. source_chem_food.txt = contains distributions for modeling variables that depend on both the source and the specific chemical. The recognized distr ibutional forms are Bernoulli, binomial, beta, discrete, empirical, exponential, gamma, lognormal, normal, point, probabilistic, and triangle. These divide into two categories. The ones that return a set of discrete values are Bernoulli, binomial, discrete , empirical, point, and probabilistic. 118 The continuous distributions are beta, exponential, gamma, lognormal, normal, point, In this study, concentration distributions for each food were obta ined from Chapter 4. 4. source_scen_food.txt. = chemicals sources to be included in the run and the scenarios to be run for each. SHEDS currently has nine scenarios (see Table 1), each of which has a column on the s ource _ Scen _food file. The first column conta ins the source type: one of food, product, or article. The second column is the source.ID, which is a brief label used for matching data to other input files. Each line on this file must have a unique source.ID, but otherwise they are arbitrary, and the us er may add new ones. The third column is a description of the source. This is not used by the model (the source.ID is always used internally). The fourth column is a flag indicating whether the source in considered to be indoors (1) or outdoors (0), which may impact the inhalation. There is one column for each exposure scenario. The source type must match the first part of the scenario name. are the combination of source and scenario will not be evaluated . Dietary exposures in SHEDS - HT are calculated by determining the total daily mass of the compound intake for each simulated person via different foods (Eqn. 5.1). For each simulated individual, (one for each food item). Dietary exposures are calculated as the sum (over all UPFs listed in the database) of the product of concentration and mass of food consumed (assigned food dia ry for the person). 119 (Eqn. 5.1) (Eqn. 5.2) (Eqn. 5.3) 5.5.2.2 Population Module A population of many thousands of individuals can be handled by the model . I nput data was based on the U.S. Census and was used to generate a simulated population representative of the U.S. population. 5.5.2.3 Exposure Assessment Test 2 nd S tage Baby Foods COP values of 12 baby foods (from BF1 - RTE to BF12 - RTE) were obtained from the study results showed in Chapter 4 (Table 4 - 3). Additional information regarding baby food samples such as brand, flavors, and serving sizes can be found in APPENDIX C. Daily intake values for each baby food were obtained from the What We Eat in America ( WWEIA ) food commodity database ((EPA), 2018) . Chemical properties such as water solubility , molecular weight , and absorption factor for each COP analyzed were obtained from PubChem.com (NIH, 2021) and the Human Metabolome Database (TMIC, 2021) . RStudio (version 1.4.1106 © 2009 - 2021) was used to run SHEDS - HT. Results showed that i nfants (6 - 12 months) could be exposed to 0.0031 mg/kg/6 mo. (Table 5 - 1) It is worth mentioning that our results could be underestimating the exposure 120 level reported in Table 5 - 1 since the intake rate of infants (6 - 12 months old) for various baby food combination meals obtained from the WWEIA database w as not available. No specific dietary value was available for each individual baby food type and mixture meals. Therefore, average values of single - main ingredients. Table 5 - 1 : Unpublished results of COPs exposure in infant (6 - 12 months old) Total COPs Exposure using SHEDS - HT 50% Percentile 95% Percentile Exposure 3 months (mg/kg/day) Exposure 6 months (mg/kg/day) Exposure 3 months (mg/kg/day) Exposure 6 months (mg/kg/day) 0.000047633 0.000095265 0.001527069 0.003054138 5.6 CONCLUSIONS Lipid oxidation species (such as COPs and POPs) are a new health and nutrition issue that requires urgent action. These compounds are promoted by different processing conditions currently employed in the food industry. Their association with several chroni c diseases and their adverse biological effects that negatively impacts human health was covered in Chapters 1 and 2. These chapters pointed the importance of considering these compounds as part of food safety processes. Additionally, it marked the need of more studies that can clarify the lack of knowledge of the occurrence of these compounds, specifically, in the Western diet. UPFs nutritional quality, specially, unavoidable changes in macronutrients quantities resulting from the heterogeneity of the man ufacturing processes, were covered in Chapter 3. This chapter can provide to the reader with nutritional and non - nutritional information regarding the effects of concurrent consumption of UPFs and, hopefully, will help the food 121 industry, policy makers, and US population to modify dietary recommendations leaning towards a healthier and chemically safe diet. It is critical to obtain the support of law makers that will ensure the creation of new regulations that will considerate these lipid oxidation species a s hazardous for human health. Information provided in Chapter 4 is a valuable tool for future studies that will elucidate the current knowledge of these species and their exact mechanism in the human body. An ADME study is essential for next research steps and it is scheduled as part of this exposure assessment. This study has a direct impact in the areas of public health, food safety, nutrition quality, and food law and ma rket (Chapter 5). Moreover, results from an exposure assessment test showed that a n infant could be ingesting a minimum of 2.86 ug per serving by consuming just 12 baby food items studied in this project . Th e purpose of the addition of ingredients to baby However, these oxidative compounds have not been fully analyzed in infants from a toxicological point of view. SHEDS - HT m odel shows that a n infant could be ingesting 0. 0031 mg/kg/6 mo. Since there is assumption could be done to determine if it should be a health risk. Once again, an ADME study and a risk assessment are imperative to understand the effect of t hese toxic compounds on infants. 5.7 FUTURE WORK An ADME study was established in Chapter 4 as the next step to address COPs as a potential health threat. Critical values/rates of absorption, deposition, metabolism, and 122 excretion of individual COPs can now be determined together with the COPs values obtained in this project. This information is vital to expand the current knowledge of COPs on metabolism (including impacts on the microbiome). In addition, once the absorbance values are obtained from the toxicolo gical analysis, they can be used to perform an exposure assessment that will provide insight of the level of exposure of COPs in the US population, and eventually, a risk assessment. 123 APPENDI CES 124 APPENDIX A TH E ROLE OF CHOLESTEROL OXIDATION PRODUCTS IN FOOD TOXICITY (JOURNAL PAPER) 125 Figure 6 : T h e R ole of C holesterol O xidation P roducts in Food Toxicity ( J ournal P aper ) 126 F igure 6 : (cont d) 127 F igure 6 : (cont d ) 128 F igure 6 : (cont d ) 129 F igure 6 : (cont d ) 130 F igure 6 : (cont d ) 131 F igure 6 : (cont d ) 132 F igure 6 : (cont d ) 133 F igure 6 : (cont d ) 134 F igure 6 : (cont d ) 135 F igure 6 : (cont d ) 136 F igure 6 : (cont d ) 137 F igure 6 : (cont d ) 138 F igure 6: (cont d ) 139 F igure 6 : (cont d ) 140 F igure 6 : (cont d ) 141 F igure 6 : (cont d ) 142 F igure 6 : (cont d ) 143 F igure 6 : (cont d ) 144 F igure 6 : (cont d ) 145 F igure 6 : (cont d ) 146 F igure 6 : (cont d ) 147 F igure 6 : (cont d ) 148 F igure 6 : (cont d ) 149 F igure 6 : (cont d ) 150 F igure 6 : (cont d ) 151 F igure 6 : (cont d ) 152 F igure 6 : (cont d ) 153 F igure 6 : (cont d ) 154 F igure 6 : (cont d ) 155 F igure 6 : (cont d ) 156 F igure 6 : (cont d ) 157 F igure 6 : (cont d ) 158 APPENDIX B PHYTOSTEROLS AND THEIR OXIDATIVE PRODUCTS IN INFANT FORMULA (JOURNAL PAPER) 159 Figure 7 : P hytosterols and their O xidative P roducts in I nfant F ormula ( J ournal P aper ) 160 Figure 7 161 Figure 7 ) 162 Figure 7 ) 163 Figure 7 ) 164 Figure 7 ) 165 Figure 7 ) 166 Figure 7 ) 167 Figure 7 ) 168 Figure 7 ) 169 Figure 7 ) 170 Figure 7 ) 171 Figure 7 ) 172 Figure 7 ) 173 Figure 7 ) 174 Figure 7 ) 175 Figure 7 ) 176 Figure 7 ) 177 Figure 7 ) 178 Figure 7 ) 179 Figure 7 ) 180 Figure 7 ) 181 Figure 7 ) 182 Figure 7 ) 183 Figure 7 ) 184 Figure 7 ) 185 Figure 7 ) 186 Figure 7 ) 187 Figure 7 ) 188 Figure 7 ) 189 Figure 7 ) 190 Figure 7 ) 191 Figure 7 ) 192 Figure 7 ) 193 Figure 7 ) 194 Figure 7 ) 195 Figure 7 ) 196 Figure 7 ) 197 Figure 7 ) 198 Figure 7 ) 199 Figure 7 ) 200 Figure 7 ) 201 Figure 7 ) 202 Figure 7 ) 203 Figure 7 ) 204 Figure 7 ) 205 APPENDIX C LIST OF ALL FOOD MEALS, THEIR TEST CODES, AND FOOD GROUP 206 Table 6 : Processed and Ultra - processed meals ID used in this study Ready to Eat (RTE) Dairy Sample ID Ultra - processed Foods (UPF) D1 - RTE American Cheese Happy Farms D2 - RTE Cheddar cheese Happy Farms D3 - RTE Margarine (regular, not low - fat, salted) Countryside Creamery *D4 RTE Butter Praire Farms D5 - RTE Cream (half & half) - Meijer D6 - RTE Swiss cheese - Kroger D7 - RTE Cream cheese Happy Farms D8 - RTE Ice cream (regular, not low - fat, vanilla) - Purple Cow D9 - RTE Yogurt (low - fat, fruit flavored) - Yoplait D10 - RTE Chocolate milk - Nesquick D11 - RTE Infant formula Little Journey Meat & Poultry MP1 - RTE Bologna - Eckrich MP2 - RTE Salami Oscar Mayer MP3 - RTE Soup bean w/bacon/pork (canned, prepared w/water) - Campbell's MP4 RTE Chili con carne w/beans (canned) - Campbell's MP5 RTE Lasagna w/meat (frozen, heated) MP6 RTE Chicken noodle soup - Kroger MP7 RTE Beef and vegetables soup Kroger MP8 RTE Mini Ravioli - Chef Boyardee MP9 - RTE Spaghetti - Chef Boyardee Seafood S1 - RTE Clam chowder (New England, canned, prep w/ whole milk) Kroger Eggs & egg derivatives E1 - RTE Mayonnaise (regular, bottled) E2 - RTE Macaroni salad (from grocery/deli) - Meijer Baby food BF1 - RTE Baby food - beef and broth/gravy Beech Nut BF2 - RTE Baby food chicken and broth/gravy - Gerber BF3 - RTE Baby food - vegetables and beef - Gerber BF4 - RTE Baby food - vegetables and chicken - Gerber BF5 - RTE Baby food - chicken noodle dinner - Gerber BF6 - RTE Baby food - macaroni, tomato and cheese - Gerber BF7 - RTE Baby food - turkey and rice - Gerber BF8 - RTE Baby food turkey and broth/gravy Beech Nut BF9 - RTE Baby food fruit yogurt - Gerber BF10 - RTE Baby food chicken with rice - Gerber 207 Table 6 (cont d) BF11 - RTE Baby food vegetables and turkey - Gerber BF12 - RTE Baby food macaroni and cheese with vegetables - Gerber BF13 - RTE Pasta pick - ups (cheese ravioli) - Gerber Others O1 - RTE Popcorn w/butter (microwave) - Kroger O2 - RTE Salad dressing (creamy/buttermilk type, regular) Garden O3 - RTE Macaroni & cheese (boiled) - Kraft O4 - RTE Macaroni & cheese (microwaved) - Kraft Fast Food (FF Meat & Poultry MP10 - FF Hamburger on bun MP11 - FF Chicken nuggets MP12 - FF Cheeseburger on bun MP13 - FF Steak tacos w/beans, lettuce, rice and cheese - Chipotle MP14 - FF Cheese and chicken quesadilla - Chipotle MP15 - FF Chicken burrito w/lettuce, cheese, pico - Chipotle MP16 - FF Chicken drumstick - KCF MP17 - FF Chicken wing - KFC MP18 - FF Beef w/vegetables - Panda Express MP19 - FF Chicken w/vegetables - Panda Express MP20 - FF Chicken filet - (broiled sandwich) MP21 - FF Roast beef, ham & Provolone - Jimmy Johns MP22 - FF Sliced turkey and bacon - Jimmy Johns MP23 - FF Supreme pizza - Marco's Pizza MP24 - FF Pepperoni pizza, hand tossed - Domino's Seafood S2 - FF Fish sandwich on bun S3 - FF Fried Shrimp Panda Express Others O5 - FF French - Fries O6 - FF McDonald's Biscuit - Big Breakfast O7 - FF McDonald's Hotcakes - Big Breakfast O8 - FF Biscuit - KFC O9 - FF French Fries - KFC O10 - FF Mashed potato - KFC *The only processed food included in this study. 208 APPENDIX D CONDITIONS OF THE COLLECTION AND PREPARATION PROCESS OF READY - TO - EAT ( RTE ) ITEMS 209 Table 7 : Detailed information for the collection and preparation process of RTE items in this analysis. Sample Sample Collection Cooking Conditions Lipid Extraction American cheese Happy Farms Local Grocery Store NR Entire Product Used Cheddar cheese Happy Farms NR Bologna Eckrich NR Salami Oscar Mayer NR Chili con carne w/beans (canned) - Campbell's Microwave: Covered, on high heat for 2 ½ to 3 min. Swiss cheese Kroger NR Cream cheese Happy Farms NR Butter Praire Farms NR Margarine (regular, not low - fat, salted) Countryside Creamery NR Mayonnaise (regular, bottled) NR Cream (half & half) - Meijer NR Soup bean w/bacon/pork (canned, prepared w/water) - Campbell's Microwave: Covered on high heat for 2 ½ to 3 min. Clam chowder (New England, canned, prep w/ whole milk) Kroger Microwave: High heat for 2 min and then for 30 s at a time until cooked, stirring each time, about 3 min. Ice cream (regular, not low - fat, vanilla) - Purple Cow NR Macaroni salad (from grocery/deli) - Meijer NR Lasagna w/meat (frozen, heated) Michael Oven bake: 25 - 35 min at 400 F. Popcorn w/butter (microwave) - Kroger Microwave: 2 2 ½ min on high heat. Ranch salad dressing (creamy/buttermilk type, regular) NR Macaroni & cheese (boiled) - Kraft Boil pan: Stir for 7 to 8 min in boiling water. 210 Tab l e 7 (c o Sample Sample Collection Cooking Conditions Lipid Extraction Macaroni & cheese (microwaved) Kraft Local Grocery Store Microwave: Uncovered, on high heat 8 to 10 min or until water is absorbed, stirring every 3 min. Entire Product Used Chicken noodle soup - Kroger Microwave: Covered, on high heat for 4 to 5 min or until hot. Beef and vegetables soup Microwave: Covered, on high heat for 4 to 5 min or until hot. Baby food - beef and broth/gravy Beech Nut NR Baby food chicken and broth/gravy - Gerber NR Baby food - vegetables and beef - Gerber NR Baby food - vegetables and chicken - Gerber NR Baby food - chicken noodle dinner - Gerber NR Baby food - macaroni, tomato, and cheese - Gerber NR Baby food - turkey and rice - Gerber NR Yogurt (low - fat, fruit flavored) - Yoplait NR Baby food turkey and broth/gravy Beech Nut NR Baby food fruit yogurt Gerber NR Baby food chicken with rice - Gerber NR Baby food vegetables and turkey - Gerber NR Baby food macaroni and cheese with vegetables - Gerber NR Infant Formula - Little Journey Mix 1 scoop of powder for every 2 ounces of water. Chocolate milk Nesquick* NR Mini Ravioli - Chef Boyardee Microwave: 1 minute 30 seconds or until warm on high heat. Spaghetti & Meatballs - Chef Boyardee Microwave: 1 minute 30 seconds or until warm on high heat. Pasta pick - ups (cheese ravioli) - Gerber NR 211 APPENDIX E CONDITIONS OF THE COLLECTION AND PREPARATION PROCESS OF FAST - FOOD (FF) MEALS 212 Table 8 : Detailed information for the collection and preparation process of RTE items in this analysis. Sample Sample Collection Lipid Extraction Quarter - pound hamburger on bun Entire product except bun Chicken nuggets Entire product used French - Fries Entire product used Quarter - pound cheeseburger on bun Entire product except bun Fish sandwich on bun Entire product except bun Biscuit - McDonald's Big Breakfast Entire product used Hotcakes - McDonald's Big Breakfast Entire product used Taco w/beef, beans, lettuce, rice, and cheese Chipotle Entire product except tortilla Cheese and chicken quesadilla Entire product used Steak burrito w/lettuce, cheese, pico Entire product except tortilla Chicken drumstick (original recipe) KFC Entire product except bones Chicken wing (original recipe) Entire product except bones Biscuit Entire product used French Fries Entire product used Mashed potato Entire product used Beef w/vegetables Panda Express Entire product used Chicken w/vegetables Entire product used Fried Shrimp Entire product used Chicken filet (broiled sandwich) Entire product except bun Roast beef, ham & Provolone Jimmy Johns Entire product except bread Sliced turkey and bacon Entire product except bread Supreme pizza Marco's Pizza Entire product used Pepperoni and cheese pizza Entire product used 213 APPENDIX F STEROLS AND PHYTOSTEROL S CONTENT IN READY - TO - EAT (RTE) AND FAST - FOOD (FF) MEALS 214 Table 9 : Unpublished results of the s terols and phytosterols content in RTE and FF meals in UPFs Ready to Eat Food Category Food Item Brassicasterol Campesterol Stigmasterol - Sitosterol Fucosterol - Tocopherol (mg/100 g fat) STD Dairy D1 - RTE ND 26.01 9.31 20.30 6.01 86.64 12.43 ND ND D2 - RTE ND ND ND ND ND ND D3 - RTE ND 33.31 14.70 43.37 12.51 136.05 5.72 ND ND D4 - RTE ND ND ND ND ND ND D5 - RTE ND ND ND ND ND ND D6 - RTE ND ND ND ND ND ND D7 - RTE ND ND ND ND ND ND D8 - RTE ND ND ND ND ND ND D9 - RTE ND ND ND ND ND ND D10 - RTE ND ND ND ND ND ND D11 - RTE ND 18.19 4.86 11.64 5.32 95.02 24.75 ND ND Meat & Poultry MP1 - RTE ND ND ND ND ND ND MP2 - RTE ND ND ND tr ND ND MP3 - RTE ND 68.45 13.99 259.84 40.55 626.48 57.01 58.18 26.67 ND MP4 - RTE ND ND 31.84 9.45 102.34 29.25 ND ND MP5 - RTE ND 14.60 5.20 ND ND 0.033 0.026 ND MP6 - RTE ND ND ND ND ND ND MP7 - RTE ND ND ND ND ND ND MP8 - RTE ND 36.47 24.74 tr 83.75 43.50 ND ND MP9 - RTE ND ND ND ND ND ND Seafood S1 - RTE ND 33.99 26.75 tr 51.87 37.21 ND ND Eggs & derivatives E1 - RTE ND 61.64 24.67 42.06 13.92 176.90 18.66 ND ND E2 - RTE ND 53.46 6.89 37.17 9.46 162.59 24.23 ND ND Baby foods BF1 - RTE ND ND ND ND ND ND BF2 - RTE ND ND ND ND ND ND BF3 - RTE ND 10.48 5.14 6.52 1.87 47.10 13.21 ND ND BF4 - RTE ND 182.76 53.20 75.16 41.86 498.98 129.71 ND ND 215 Table 9 (cont BF5 - RTE ND 136.48 47.29 33.77 7.19 391.77 93.78 ND ND BF6 - RTE ND 21.98 6.84 ND 100.31 19.76 ND ND BF7 - RTE ND 190.88 21.15 ND 421.59 137.27 ND ND BF8 - RTE ND ND ND ND ND ND BF9 - RTE ND ND ND ND ND ND BF10 - RTE ND 48.06 9.69 25.41 14.33 160.14 26.56 ND ND BF11 - RTE 19.72 3.21 ND 48.31 2.23 435.62 41.06 ND ND BF12 - RTE 29.60 3.30 99.33 24.73 ND 236.18 18.19 ND ND BF13 - RTE ND 44.86 3.55 ND ND 0.046 0.014 ND Others O1 - RTE ND 34.54 5.32 ND ND ND ND O2 - RTE ND 50.87 14.28 38.09 10.27 148.17 8.02 ND 0.019 0.012 O3 - RTE ND 38.65 19.12 ND ND 5.45 1.58 ND O4 - RTE ND 26.01 9.31 16.34 9.22 124.15 12.47 39.85 17.26 ND Fast Food Food Category Food Item Brassicasterol Campesterol Stigmasterol - Sitosterol Fucosterol - Tocopherol (mg/100 g fat) STD Meat & Poultry MP10 - FF ND ND ND ND ND ND MP11 - FF ND 122.85 18.26 ND 296.67 38.94 ND ND MP12 - FF ND ND ND ND ND ND MP13 - FF ND 66.46 14.08 26.36 10.01 176.24 26.14 ND ND MP14 - FF ND 81.36 13.67 209.33 40.88 ND ND ND MP15 - FF ND ND 53.03 7.46 ND ND ND MP16 - FF ND 86.20 33.23 ND 165.55 63.66 ND ND MP17 - FF 27.56 4.66 101.79 8.40 ND 173.08 17.49 ND ND MP18 - FF ND 68.98 23.39 48.25 13.69 343.35 137.26 ND ND MP19 - FF ND 4.77 3.44 ND 33.40 17.52 ND ND MP20 - FF ND 22.44 7.49 8.93 7.17 95.24 9.40 ND ND MP21 - FF ND ND ND 39.39 6.79 ND ND 216 Table 9 (cont MP22 - FF ND 20.69 10.72 ND 34.28 4.46 ND ND MP23 - FF ND 83.95 7.74 168.10 24.33 ND ND ND MP24 - FF ND 23.57 7.87 ND 68.94 6.79 ND ND Seafood S2 - FF ND 60.10 22.86 ND 199.39 26.35 ND ND S3 - FF ND 25.42 4.88 19.73 13.05 36.94 4.58 ND ND Others O5 - FF ND 192.00 21.55 ND 331.87 34.11 ND ND O6 - FF ND ND 36.45 17.21 ND ND ND O7 - FF ND 40.86 5.16 224.87 30.79 ND ND ND O8 - FF ND 35.93 23.68 ND 74.36 8.20 ND ND O9 - FF 44.19 17.67 184.08 25.17 ND 333.41 45.98 ND ND O10 - FF ND 60.98 12.72 ND 102.64 19.30 tr ND 217 LITERATURE CITED 218 LITERATURE CITED (EPA), E. 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