Article pubs.acs.org/JAFC
Formation of Malondialdehyde, 4‑Hydroxynonenal, and 4‑Hydroxyhexenal during in Vitro Digestion of Cooked Beef, Pork, Chicken, and Salmon Christina Steppeler,†,†,‡ John-Erik Haugen,‡ Rune Rødbotten,‡ and Bente Kirkhus‡ †
Norwegian University of Life Sciences, Department of Food Safety and Infection Biology, P.O. Box 8146, Dep, 0033 Oslo, Norway Nofima, Norwegian Institute of Food, Fisheries and Aquaculture Research, Osloveien 1, 1430 Ås, Norway
‡
ABSTRACT: Red meat high in heme iron may promote the formation of potentially genotoxic aldehydes during lipid peroxidation in the gastrointestinal tract. In this study, the formation of malondialdehyde (MDA) equivalents measured by the thiobarbituric acid reactive substances (TBARS) method was determined during in vitro digestion of cooked red meat (beef and pork), as well as white meat (chicken) and fish (salmon), whereas analysis of 4-hydroxyhexenal (HHE) and 4-hydroxynonenal (HNE) was performed during in vitro digestion of cooked beef and salmon. Comparing products with similar fat contents indicated that the amount of unsaturated fat and not total iron content was the dominating factor influencing the formation of aldehydes. It was also shown that increasing fat content in beef products caused increasing concentrations of MDA equivalents. The highest levels, however, were found in minced beef with added fish oil high in unsaturated fat. This study indicates that when ingested alone, red meat products low in unsaturated fat and low in total fat content contribute to relatively low levels of potentially genotoxic aldehydes in the gastrointestinal tract. KEYWORDS: red meat, salmon, malondialdehyde, TBARS, 4-hydroxynonenal, 4-hydroxyhexenal, in vitro digestion, lipid peroxidation
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INTRODUCTION Intake of red meat (beef, pork, lamb, and goat) and processed meat is associated with increased risk of colorectal cancer (CRC).1,2 Both epidemiologic3,4 and experimental evidence5,6 indicate that the high level of heme iron in red meat may contribute to the promotion of CRC. Besides acting as a catalyst that facilitates the endogenous formation of potentially carcinogenic N-nitroso compounds (NOCs),7 heme iron may provoke carcinogenesis through the formation of cytotoxic and genotoxic aldehydes by lipid peroxidation.8 Several studies have monitored the oxidation occurring during digestion of meat in the gastric phase9−11 and identified the stomach as a bioreactor where an acidic and oxygen-rich environment promotes peroxidation.12 One of the most abundant secondary oxidation products and commonly used biomarker for oxidative stress is malondialdehyde (MDA), which is formed during the decomposition of lipid hydroperoxides produced during peroxidation of unsaturated fatty acids, preferentially long-chain polyunsaturated fatty acids.9,13,14 The mutagenic and genotoxic properties of MDA have been extensively described.15 Other potentially genotoxic secondary oxidation products are 4-hydroxynonenal (4-HNE) and 4hydroxyhexenal (4-HHE), which are products of the oxidative breakdown of hydroperoxides derived from n-6 and n-3 polyunsaturated fatty acids, respectively. Next to MDA, they represent the major aldehydes formed during peroxidation.16−18 In contrast to other biomarkers of oxidative stress in vivo, such as the isoprostanes, the levels of MDA and 4hydroxyalkenals in blood and urine are highly affected by dietary factors, for example, peroxidation during digestion.8 In particular, MDA and 4-HNE has been shown to be involved in a number of pathologies such as metabolic diseases, neuro© XXXX American Chemical Society
degenerative diseases, and cancers, probably due to their chemical reactivity and ability to form covalent adducts with macromolecules.19 High levels of MDA and 4-HNE in serum, urine and feces have been associated with various types of cancers, including cancers in the digestive tract.8,20−22 The present study investigates the formation of 4-HNE and 4-HHE and MDA equivalents measured as TBARS (thiobarbituric acid reactive substances) during in vitro digestion of fish and meat products commonly used for domestic cooking. Since the formation of these aldehydes during digestion may contribute to an increased risk of CRC, it is hypothesized that red meat (beef and pork) due to a high content of heme iron may induce higher levels of aldehydes than white meat (chicken) and fish (salmon), which are not associated with CRC. Since differences in total fat content and fatty acid composition may influence the rate of peroxidation in the gastrointestinal tract, beef products with increasing fat contents and a beef product with added fish oil were included in the study. To our knowledge, a comparative study of gastrointestinal peroxidation of marine, mammalian, and avian protein sources has not been reported before.
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MATERIALS AND METHODS
Chemicals. Pepsin (porcine, P7000, 683 U/mg solid), pancreatin (porcine, P1750), bile extract (bovine/ovine, B8381), thiobarbituric acid (TBA), 1,1,3,3-tetraethoxypropane (TEP), propyl gallate, and ethylenediaminetetraacetic acid (EDTA) were obtained from SigmaAldrich Co (St. Louis, MO). Trichloroacetic acid (TCA) was Received: August 27, 2015 Revised: December 10, 2015 Accepted: December 13, 2015
A
DOI: 10.1021/acs.jafc.5b04201 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 1. Contents of Fat, Nitrite, Iron, and Peroxide Value (PV) in Raw Materials study 1
a
study 2
sample
minced beef
minced pork
minced chicken
salmon loin
beef sirloin
minced beef
minced beef
minced beef + fish oil
fat, declared (%) fat, analyzed (%) iron (mg/kg) pv (mequiv/kg fat) nitrite (mg/kg)
10.0 8.9 20.0
