Retardation of 4-Hydroxy-2-Transnonenal (HNE), a Toxic Aldehyde Formation by Antioxidants in Heat-Treated Corn Oil at Frying Temperature

The antioxidative properties of four antioxidants such as rosemary extracts (RE), tert-butylhydroquinone (TBHQ), ascorbyl palmitate (AP), citric acid (CA) and their mixtures were investigated on the formation of 4-hydroxy-2-transnonenal (HNE) in commercial corn oil heated at 185˚C for up to 6 hours. Among the antioxidants 100 ppm RE and a mixture of 200 ppm tertiary butylated hydroquinone (TBHQ) + 100 ppm ascorbyl palmitate (AP) + 50 ppm citric acid (CA) exhibited excellent antioxidative activity, as determined by the thiobarbituric acid reaction (TBARS) assay, measuring the formations of the secondary lipid oxidation products and by high-performance liquid chromatography (HPLC), measuring the formation of the toxic α, β-unsaturated hydroxyaldehyde HNE after heat treatment of corn oil at 185˚C up to 6 hours. TBHQ, AP and CA alone did not show much protective properties. The synergistic effects of TBHQ + AP + CA mixture shown to reduce the formation of HNE after 6 hours heat-treated corn oil by 27%. RE 100 ppm was also found to be a very effective antioxidant, reducing the formation of HNE after 6 hours heat-treated corn oil in the same condition by


Introduction
Lipid peroxidation of polyunsaturated fatty acids (PUFA) leads to a large variety How to cite this paper: Jin, W., Shoeman, D.W., Shukla, V.K.S. and Csallany, A.S. (2020) Retardation of 4-Hydroxy-2-Transnonenal (HNE), a Toxic Aldehyde Formation by Antioxidants in Heat-Treated Corn Oil at of secondary lipid peroxidation products, including the α, β-unsaturated hydroxyaldehydes such as the toxic aldehyde 4-hydroxy-2-transnonenal (HNE) [1] [2] [3]. This polar very reactive aldehyde containing unsaturation between the α and β carbons and has a hydroxyl group on the carbon 4 position. The chemical structure makes this aldehyde very reactive to amino, sulfhydryl and thiol groups and therefore reacts aggressively with biological compounds. Among the four α, β-unsaturated hydroxyaldehydes (HHE, HOE, HNE, HDE), the 9 carbon containing HNE was found to be the most reactive and toxic compound [4]- [12].
Its toxicity has been demonstrated in the literature by its reaction to DNA and RNA in low concentrations and related to a number of pathological conditions including inflammatory and degenerative processes such as atherosclerosis, liver damage, Parkinson's, Alzheimer's and other diseases [13]- [19].
It has been shown that the precursor of HNE is linoleic acid [20] and corn oil together with soybean oil contain high levels of this fatty acid. Corn oil contains about 60% linoleic acid and is commonly used for frying in the food industry, frying fast foods commercially and used also in households for frying. A number of previous experiments have shown the formation of HNE due to heat treatments in the above oils by this laboratory [20]- [25]. It is known that certain antioxidants can delay the reactions of lipid peroxidation and therefore could cause some protection to heat-treated PUFA oils, and lower the formation of secondary lipid oxidation products. HNE, a toxic secondary lipid oxidation product, was shown to form linoleic acid due to heat treatment at frying temperatures and it has also been shown to incorporate into fried food [26] [27]. Since HNE has been shown to be absorbed from the diet and metabolized [28], it is important to investigate how its formation can be reduced during heat treatments at frying temperature. The objective of the present study was to measure the antioxidant effects of several synthetic and a naturally occurring antioxidant, rosemary [29] and measure the lowering effects of the formation of the most toxic and abundant α, β-unsaturated hydroxyaldehyde isomer, HNE in heat-treated corn oil which is high in linoleic acid, a precursor for HNE.

Chemicals and Instruments
Mazola corn oil was purchased from a local store (Roseville, MN

Methods
The detailed methods used in the present experiments was described by Seppanen and Csallany [21] [22] and recently by Juan, Shoeman and Csallany [25].
Stock Solutions in 10 mL Ethanol:
Brief description of the method: 1) The preparation of 2,4-dinitrophenylhydrazine (DNPH) reagent was made from 10 mg DNPH, three times recrystallized from methanol. The recrystallized DNPH was mixed with 20 mL 1N HCl and heated at 50˚C for 1 h. After cooling it was extracted four times with HPLC-grade hexane to remove impurities. The DNPH reagent was used immediately.
2) Transesterification of aldehydes and related carbonyl compounds was carried out in duplicates by using 1 gr of unheated or previously heated oil samples, and 5 mL of freshly prepared DNPH reagents, the samples were shaken overnight at room temperature in the dark.
3) The DNPH derivatives were extracted from the oil with 10 mL of methanol:water (75:25, v/v) repeatedly three times.
4) From the combined methanol:water samples, the DNPH derivatives were extracted three times with 10 mL of dichloromethane. The combined dichloromethane extracts were concentrated under N 2 gas to 0.5 mL.
5) The DNPH derivatives, from the 0.5 mL dichloromethane extracts, were separated to polar and nonpolar compounds and osozones by thin-layer chromatography (TLC) using 2 silica gel plates per sample and developed in dichloromethane.
6) The polar compounds were individually eluted from the thin plates 3 times with 10 mL methanol. The combined methanol extracts, after centrifugation, to remove residual silica gel, were concentrated under N 2 gas to 1 mL. The concentrated polar DNPH derivatives were filtered by a syringe filter nylon membrane Known amount of sample was mixed with known amount of the pure standard.
The recovery was calculated from the increased peak area due to the added standard. Recovery rates were between 95% and 105%. The detection limit of samples was 1 ng. Since every duplicate sample was analyzed separately and injected in duplicates for HPLC analysis, the minimum numbers of the HPLC chromatograms were 4 for each sample analyzed.

Fatty Acid Distribution
The fatty acid distribution of the unheated corn oil was measured by gas chromatography using the method of Metcalf and Schmitz [31].

Statistical Analysis
Analysis of variance was used to determine the significant differences between groups. All measurements were replicated three times. The results obtained were statistically analyzed with two-way ANOVA. The Tukey test was conducted to calculate P values. Significant differences were determined at P ≤ 0.05.

Results and Discussion
The main objective of the present experiments was to measure the heat-induced secondary lipid peroxidation, with special reference to the formations of the toxic 4-hydroxynonenal (HNE) in the presence and absence of certain antioxidants.
Before the major objective, to measure the formation of HNE, preliminary experiments were conducted including the TBARS assay to measure the formation of all aldehydes and related carbonyl compounds at 185˚C frying temperature, up to 6 hours, in the presence and absence of certain antioxidants [33].  [20].   ppm, can significantly lower the oxidation, at frying temperature, the 6 hour heat treated corn oil. A previous study reported that AP has antioxidant activity [34]. It was also reported that AP retarded the oxidation in oil at frying temperature [35]. However, in the present study, AP alone had no lowering effects of TBARS formation of heat-treated corn oil.     properties are mainly phenolic diterpenes, such as carnosol, carnosic acid, rosmanol, epirosmanol and isorosmanol [28].
The deterioration of frying fats and oils at high temperatures is complicated, because oxidative and thermolytic reactions are occurring simultaneously, and both the saturated and unsaturated fatty acids undergo chemical degradations when exposed to high temperatures and oxygen [38] resulting in the formation of aldehydes, ketones and related carbonyl compounds.   However, the protective effect was not due to antioxidants, but the protection was due to the prevention of oxygen penetration into the monolayer of the oil surface, therefore lowering the oxidation of the oil and lowering the decomposition of linoleate and the formation of HNE [39].     In summary, the greatest reduction of HNE formation (29%) after 6 hours of heat treatment at 185˚C was produced by the addition of 100 ppm RE. This was followed by the suppression of 27% HNE by the combination of 200 ppm TBHQ + 100 ppm AP + 50 ppm CA.
In conclusion, the present experiments demonstrated that certain synthetic antioxidant mixtures and a naturally occurring antioxidant at certain low concentrations will retard the overall lipid peroxidation process of heat-treated commercial corn oil at of 185˚C up to 6 hours as measured by the TBARS assay and also reduce the formation of the most toxic α, β-unsaturated hydroxyaldehyde HNE, measured by HPLC.