Hydrogenation

Tritiated RvE1 (6,7,14,15-[³H]RvE1; ∼100 Ci/mmol) was obtained by custom catalytic hydrogenation of synthetic 6,14-diacetylenic RvE1 methyl ester (Fig. 2 a, compound 4) that was supplied to and performed at American Radiolabeled Chemicals, and the ³H-labeled RvE1 was saponified and isolated by HPLC. [³H]-RvE1–specific binding was performed with CHO cells (American Type Culture Collection) transfected with human recombinant ChemR23. Cells were suspended in Dulbecco's phosphate buffered saline with CaCl₂ and MgCl₂ (DPBS²⁺). For saturation binding, aliquots (10⁶ cells) were incubated with increasing concentrations of [³H]-RvE1 in the presence or absence of unlabeled homoligand (10 μM) for 1 h at 14°C. To determine ligand specificity by competition binding, aliquots (10⁶ cells) were incubated with 10 nM of [³H]-RvE1 in the presence of various competitors (10 μM) for 1 h at 14°C. Chemerin COOH-terminal bioactive peptide (YHSFFFPGQFAFS) was synthesized by SynPep Co. as described previously (17 (link)). The bound and unbound radioligands were separated by filtration through Whatman GF/C glass microfiber filters (Fisher Scientific) and radioactivity was determined. Scatchard plot was obtained and Kd and Bmax values were calculated using Prism (Graphpad Software, Inc.).

Reported single food items and the underlying ingredients of culinary preparations (handmade recipes) were classified according to NOVA food classification system into the following four groups (and subgroups within these groups): group 1—unprocessed or minimally processed foods (eg, rice and other cereals, meat, fish, milk, eggs, fruit, roots and tubers, vegetables, nuts and seeds); group 2—processed culinary ingredients (eg, sugar, plant oils and butter); group 3—processed foods (eg, processed breads and cheese, canned fruit and fish, and salted and smoked meats); group 4—ultra-processed foods (eg, confectioneries, savoury snacks, fast food dishes, mass-produced packaged breads, frozen and ready meals and soft drinks).10 12 (link)
Ultra-processed foods, which are the focus of interest in this study, as previously mentioned, are formulations of low-cost ingredients, many of non-culinary use, that result from a sequence of industrial processes. Processes underlying the manufacture of ultra-processed foods start with the extraction of substances existing in intact foods, such as oils, fats, sugars, starches and protein. Intermediate processes may involve hydrolysis, hydrogenation and other chemical modifications of the extracted substances. Other steps include the assembling of modified (eg, hydrogenated oils) and unmodified (eg, sugar) substances using processes such as extrusion and prefrying, the addition of additives and sophisticated packaging with the frequent employment of novel synthetic materials.10 12 (link)
Food items were ultimately classified as ultra-processed if they contained ingredients found exclusively in these products. These ingredients are substances derived from foods but of non-culinary use (eg, protein isolates, modified starches, hydrogenated or interesterified oils) and classes of additives with cosmetic functions (eg, colourants, flavourings, artificial sweeteners, emulsifiers, thickeners and bleaching, bulking, firming, gelling, glazing, foaming and carbonating agents). The presence or absence of these ingredients was identified from auxiliary AUSNUT data sources (Food details file and Food recipe file)21 and from list of ingredients obtained from food packages or from company websites (see online supplementary appendix 1 to 2). More information regarding how to identify ultra-processed foods can be found elsewhere.10 12 (link)
For all food items judged to be a culinary preparation, the recipes were disaggregated using the AUSNUT 2011–2013 Food Recipe File,21 enabling the classification of composite foods into all NOVA food groups. A total of 2585 (45%) food codes were subject to disaggregation, and this process was continued until all ingredients were single food items.
To classify all food items, two experts with Australian food and dietary intake knowledge applied the NOVA system to the AUSNUT 2011–2013. All classifications were checked by another two independent food assessment experts, and where classification discrepancies arose, these were discussed until consensus was reached among all researchers. The NOVA system was applied to the AUSNUT classification system that considers a major (two-digit), submajor (three-digit) or minor (five-digit) food group. The survey ID (eight-digit) assigned to each food item was used when it was not possible to discriminate the degree of food processing within a minor group (table 1).
When the classification of a food item was not clear (eg, cake or cupcake, honey, commercial or homemade), the conservative alternative was chosen (homemade in this case, and thus disaggregated). Additional procedures were applied to classify breads with generic food item descriptions based on the sampling details information comprised in the AUSNUT ‘Food details file’. Unlike other countries, many commercially produced breads in Australia are processed rather than ultra-processed, that is, their ingredients do not include neither food substances of no culinary use nor cosmetic additives. Of the 62 generic bread codes where the NOVA classification was not easily apparent, there were two generic bread codes that contributed the most to total bread energy intake (25% combined): (1) bread, from white flour, commercial; (2) bread roll, from white flour, commercial. They were classified as ultra-processed foods since the samples that composed the AUSNUT 2011–2013 were mostly of mass-produced branded breads with cosmetic additives. All the remaining infrequent breads were classified as processed as the conservative hypothesis (see details in the online supplementary appendix 1 to 2).