**Exposure of Human Gastric Cells to Oxidized Lipids Stimulates Pathways of Amino Acid Biosynthesis on a Genomic and Metabolomic Level**

**Mathias Zaunschirm 1,**† **, Marc Pignitter 1,\* ,**† **, Antonio Kopic <sup>1</sup> , Claudia Keßler <sup>1</sup> , Christina Hochkogler <sup>1</sup> , Nicole Kretschy <sup>2</sup> , Mark Manuel Somoza <sup>2</sup> and Veronika Somoza <sup>1</sup>**


Academic Editor: Severina Pacifico

Received: 15 October 2019; Accepted: 12 November 2019; Published: 14 November 2019

**Abstract:** The Western diet is characterized by a high consumption of heat-treated fats and oils. During deep-frying processes, vegetable oils are subjected to high temperatures which result in the formation of lipid peroxidation products. Dietary intake of oxidized vegetable oils has been associated with various biological effects, whereas knowledge about the effects of structurally-characterized lipid peroxidation products and their possible absorption into the body is scarce. This study investigates the impact of linoleic acid, one of the most abundant polyunsaturated fatty acids in vegetable oils, and its primary and secondary peroxidation products, 13-HpODE and hexanal, on genomic and metabolomic pathways in human gastric cells (HGT-1) in culture. The genomic and metabolomic approach was preceded by an up-to-six-hour exposure study applying 100 µM of each test compound to the apical compartment in order to quantitate the compounds' recovery at the basolateral side. Exposure of HGT-1 cells to either 100 µM linoleic acid or 100 µM 13-HpODE resulted in the formation of approximately 1 µM of the corresponding hydroxy fatty acid, 13-HODE, in the basolateral compartment, whereas a mean concentration of 0.20 ± 0.13 µM hexanal was quantitated after an equivalent application of 100 µM hexanal. An integrated genomic and metabolomic pathway analysis revealed an impact of the linoleic acid peroxidation products, 13-HpODE and hexanal, primarily on pathways related to amino acid biosynthesis (*p* < 0.05), indicating that peroxidation of linoleic acid plays an important role in the regulation of intracellular amino acid biosynthesis.

**Keywords:** linoleic acid peroxidation products; hexane; gastric cells; metabolomics; cDNA microarray

#### **1. Introduction**

In industrialized countries, the habitual diet is characterized by a high intake in dietary fats, mainly originating in heat-treated foods [1]. Among them, the popularity of deep-fried products is based on convenience, their crispy texture, and pleasant mouth feel compared to non-fried foods [2]. During the deep-frying process, the frying oils are severely heated, resulting in multiple chemical reactions of the oils' constituents [3]. One of the major determinants of the quality of a frying oil are lipid peroxidation products. Here, primary and secondary lipid peroxidation products are formed through autoxidation or photo-oxidation reactions of unsaturated fatty acids, resulting in the generation of lipid hydroperoxides and further decomposition products, such as aldehydes, carboxylic acids, alcohols, or hydrocarbons [4]. Under thermal treatment, free fatty acids undergo faster oxidation processes than

under non-heated conditions [5]. The free fatty acid, linoleic acid, is an essential unsaturated fatty acid and the predominant omega-6 polyunsaturated fatty acid in the Western diet. Linoleic acid can be found in vegetable oils commonly used for deep-frying, such as rice bran, safflower, sunflower, soybean, corn, and canola oil [6,7]. Other prominent sources of linoleic acid, and therefore prone to oxidation, are walnuts, pine nuts, and pecans [8].

In several animal feeding studies, highly oxidized fats with peroxide values >10 meq/kg oil have been administered [9–12]. On the one hand, oxidized fats and oils have been hypothesized to be harmful to health and associated with, e.g., the development of atherosclerosis [13]. On the other hand, animal feeding studies have also shown that dietary oxidized fats lead to a decrease in triacylglycerols and cholesterol in liver and plasma, and to the regulation of genes involved in lipid metabolism [14]. However, studies on the effects of structurally-characterized primary and secondary peroxidation products of linoleic acid are lacking. Moreover, it has been shown that the primary peroxidation product of linoleic acid, 13-hydroperoxy-9*Z*,11*E*-octadecadienoic acid (13-HpODE), is mainly decomposed in the stomach into secondary peroxidation products such as its corresponding alcohols, epoxyketones and aldehydes, which were demonstrated to be partially incorporated into the intestinal lumen and further absorbed by enterocytes [10,15].

Since the stomach is a very reactive environment for the chemically rather instable dietary lipid peroxidation products [16], the presented work addresses whether linoleic acid and its primary and secondary peroxidation products, 13-HpODE and hexanal, are absorbed by human gastric cells (HGT-1) and show pre-absorptive cellular effects on genomic and metabolic levels.

#### **2. Results**
