Elucidation of decomposition pathways of linoleic acid hydroperoxide isomers by GC-MS and LC-MS/MS.

Food lipid oxidation provides various volatile compounds involved in food flavor via the decomposition of lipid hydroperoxide (LOOH). This study predicted the pathways which can coherently explain LOOH decomposition focusing on hydroperoxy octadecadienoic acid (HpODE) isomers (9-EZ-HpODE, 9-EE-HpODE, 10-HpODE, 12-HpODE, 13-ZE-HpODE, and 13-EE-HpODE) which are the major LOOH contained in edible oils. Each standard was first prepared and thermally decomposed. Generated volatile and non-volatile compounds were analyzed by GC-MS and LC-MS/MS. The results showed that all HpODE decomposition was based on the factors such as favorable scission, radical delocalization, and cyclization. Interestingly, the formation of 8-HpODE and 14-HpODE were demonstrated during HpODE decomposition. The insights obtained in this study would explain the generation pathways of flavor involved in food quality.

Dietary triacylglycerol hydroperoxide is not absorbed, yet it induces the formation of other triacylglycerol hydroperoxides in the gastrointestinal tract.

The in vivo presence of triacylglycerol hydroperoxide (TGOOH), a primary oxidation product of triacylglycerol (TG), has been speculated to be involved in various diseases. Thus, considerable attention has been paid to whether dietary TGOOH is absorbed from the intestine. In this study, we performed the lymph duct-cannulation study in rats and analyzed the level of TGOOH in lymph following administration of a TG emulsion containing TGOOH. As we successfully detected TGOOH from the lymph, we hypothesized that this might be originated from the intestinal absorption of dietary TGOOH [hypothesis I] and/or the in situ formation of TGOOH [hypothesis II]. To determine the validity of these hypotheses, we then performed another cannulation study using a TG emulsion containing a deuterium-labeled TGOOH (D2-TGOOH) that is traceable in vivo. After administration of this emulsion to rats, we clearly detected unlabeled TGOOH instead of D2-TGOOH from the lymph, indicating that TGOOH is not absorbed from the intestine but is more likely to be produced in situ. By discriminating the isomeric structures of TGOOH present in lymph, we predicted the mechanism by which the intake of dietary TGOOH triggers oxidative stress (e.g., via generation of singlet oxygen) and induces in situ formation of TGOOH. The results of this study hereby provide a foothold to better understand the physiological significance of TGOOH on human health.

Elucidation of Olive Oil Oxidation Mechanisms by Analysis of Triacylglycerol Hydroperoxide Isomers Using LC-MS/MS.

Despite the importance of the insight about the oxidation mechanisms (i.e., radical and singlet oxygen (1O2) oxidation) in extra virgin olive oil (EVOO), the elucidation has been difficult due to its various triacylglycerol molecular species and complex matrix. This study tried to evaluate the mechanisms responsible for EVOO oxidation in our daily use by quantitative determination of triacylglycerol hydroperoxide (TGOOH) isomers using LC-MS/MS. The standards of dioleoyl-(hydroperoxy octadecadienoyl)-triacylglycerol and dioleoyl-(hydroperoxy octadecamonoenoyl)-triacylglycerol, which are the predominant TGOOHs contained in EVOO, were prepared. Subsequently, fresh, thermal-, and photo-oxidized EVOO were analyzed. The obtained results mostly agreed with the previously reported characteristics of the radical and 1O2 oxidation of linoleic acid and oleic acid. This suggests that the methods described in this paper should be valuable in understanding how different factors that determine the quality of EVOO (e.g., olive species, cultivation area, cultivation timing, and extraction methods) contribute to its oxidative stability.

Carboxylic acids derived from triacylglycerols that contribute to the increase in acid value during the thermal oxidation of oils.

Acid value (AV), is a widely used indicator of oil degradation that, by definition, measures the free fatty acids formed via the hydrolysis of triacyclglycerols. However, based on observations made in previous studies, we hypothesized that the oxidation of triacylglycerols leads to the formation of carboxylic acids with a glycerol backbone which are also calculated as AV. In this study, we aimed to identify such carboxylic acids and prove the above hypothesis. Heating a canola oil at 180 °C for 6 h without the addition of water resulted in an increase in AV from 0.054 to 0.241. However, the contribution of free fatty acids to this increase in AV was minimal; free fatty acid-derived AV before and after heating was 0.020 and 0.023, respectively. Then, via mass spectrometric analyses, we identified two 8-carboxy-octanoyl (azelaoyl) -triacylglycerols (i.e., dioleoyl-azelaoyl-glycerol and oleoyl-linoleoyl-azelaoyl-glycerol) in the heated oil. Azelaoyl-triacylglycerols-derived AV before and after heating the oil was 0.008 and 0.109, respectively, demonstrating that azelaoyl-triacylglycerols contribute to AV. Such an increase in AV by azelaoyl-triacylglycerols was also observed in an oil used to deep-fry potatoes (i.e., an oil with a relatively high water content). These results suggest that AV is also an indicator of the thermal oxidation of triacylglycerols.

Determination of acrolein generation pathways from linoleic acid and linolenic acid: increment by photo irradiation.

2-Propenal (acrolein) is a toxic aldehyde generated from the thermal degradation of edible oils. While previous studies have suggested that linolenic acid (LnA) is the origin of acrolein formation in edible oils, these studies were performed under thermal conditions where only the fatty acid hydroperoxide (FAOOH) isomers derived from radical oxidation were formed. In this study, we reinvestigated the acrolein generation pathway through another oxidation mechanism involving singlet oxygen (1O2) oxidation (type II photo-oxidation). Standards of the main FAOOH isomers (oleic acid hydroperoxide, linoleic acid hydroperoxide (HpODE), and linolenic acid hydroperoxide (HpOTE)) found in edible oils were prepared, and their decomposition products, including those derived from1O2 oxidation (i.e., 10- and 12-HpODE) were analyzed by GC-EI-MS. We found that 1O2 oxidation products of linoleic acid (LA) and LnA but not OA, are significant sources of acrolein formation. The amount of acrolein formed from edible oils high in LA (e.g., rice bran oil) increased by photo irradiation. Further investigation into the mechanism of acrolein generation demonstrated that the amount of acrolein derived from 1O2 oxidation-specific HpOTE isomers (i.e., 10- and 15-HpOTE) was two times greater than that of other HpOTE isomers (i.e., 9-, 12-, 13-, and 16-HpOTE). The results of the present study provide a new pathway of acrolein formation from type II photo-oxidation. This information can be used to inform on oil storage and processing conditions to reduce exposure and dietary intake of acrolein.

Structural Analysis of Lipid Hydroperoxides Using Mass Spectrometry with Alkali Metals.

Lipid oxidation is involved in various biological phenomena (e.g., oxylipin generation and oxidative stress). Of oxidized lipid structures, the hydroperoxyl group position of lipid hydroperoxides (LOOHs) is a critical factor in determining their biological roles. Despite such interest, current methods to determine hydroperoxyl group positions possess some drawbacks such as selectivity. While we previously reported mass spectrometric methods using Na+ for the highly selective determination of hydroperoxyl group positions, nothing was known except for the fact that sodiated LOOHs (mainly linoleate) provide specific fragment ions. Thus, this study was aimed to investigate the effects of different alkali metals on the fragmentation of LOOHs, assuming its further application to analysis of other complex LOOHs. From the analysis of PC 16:0/18:2;OOH (phosphatidylcholine) and FA 18:2;OOH (fatty acid), we found that fragmentation pathways and ion intensities largely depend on the binding position and type of alkali metals (i.e., Li+, Hock fragmentation; Na+ and K+, α-cleavage (Na+ > K+); Rb+ and Cs+, no fragmentation). Furthermore, we proved that this method can be applied to determine the hydroperoxyl group position of esterified lipids (e.g., phospholipids and cholesterol esters) as well as polyunsaturated fatty acids (PUFAs) including n-3, n-6, and n-9 FA. We anticipate that the insights described in this study provide additional unique insights to conventional lipid oxidation research.

Effect of Furan Fatty Acids and 3-Methyl-2,4-nonanedione on Light-Induced Off-Odor in Soybean Oil.

Soybean oil is one of the most widely consumed vegetable oils. However, under photooxidative conditions, this oil develops a beany and green off-odor through a mechanism that has not yet been elucidated. Upon photooxidation, 3-methyl-2,4-nonanedione (3-MND) produces a strong aroma. In this study, the effect of furan fatty acids and 3-MND on odor reversion in soybean oil was investigated. Our findings suggest that the observed light-induced off-odor was likely attributable to the furan fatty acids present in the oil through the generation of 3-MND. While 3-MND may not be directly responsible for the development of light-induced off-odor, this compound appears to be involved because off-odor was detected in canola oil samples containing added 3-MND. In addition, in the present work, 3-hydroxy-3-methyl-2,4-nonanedione, which is derived from 3-MND, was identified for the first time in light-exposed soybean oil and shown to be one of the compounds responsible for odor reversion.

Effects of Minor Components of Crude Vegetable Oil on the Enzymatic Method to Analyze Positional Fatty Acid Distributions in Triacylglycerols withCandida antarctica Lipase B.

Crude soybean and rapeseed oils were subjected to the method to determine FA distributions in TAG using Candida antarctica lipase B, giving similar results to those for refined oils. Minor components in crude oils, such as percentages of FFA or phospholipids were indicated not to affect 1(3)-selective transesterification by the lipase and FA compositional analysis of the resulting 2-MAG fraction significantly. Phospholipids were confirmed not to contaminate the 2-MAG fraction. Oxidized soybean oil with a PV of 10 meq/kg also gave similar results to the ones for refined oil. The method was confirmed to be applicable for crude oils and oxidized oils with a PV smaller than 10 meq/kg without prior purification of TAG.

Relationship between 3-Methyl-2,4-nonanedione Concentration and Intensity of Light-induced Off-odor in Soy Bean Oil.

A beany and green off-odor is developed in soy bean oil (SBO) under light-induced oxidative conditions. 3-Methyl-2,4-nonanedione (3-MND) was inferred as the compound responsible for the off-odor. In this study, we designed a simple quantification method for 3-MND in SBO, and evaluated the relationship between the 3-MND concentration and the intensity of the off-odor. 3-MND was analyzed by GC/MS with a thermal desorption unit system. By our method, the 3-MND concentration was found to increase with storage days and the SBO content under light exposure, and there was a high correlation between the measured 3-MND concentration and the intensity of the light-induced off-odor in SBO (R = 0.9586).

The Collaborative Study on the Enzymatic Analysis of Positional Distribution of Short- and Medium-chain Fatty Acids in Milk Fat Using Immobilized Candida antarctica Lipase B.

The positional distributions of fatty acids (FAs) in milk fat containing short- and medium-chain FAs were analyzed by sn-1(3)-selective transesterification of triacylglycerols (TAGs) with ethanol using immobilized Candida antarctica lipase B (CALB), in a collaborative study conducted by 10 laboratories. The mean C4:0, C6:0, and C8:0 FA contents, when analyzed as propyl esters (PEs) using gas chromatography (GC) with a DB-23 capillary column, were found to be 3.0, 2.0, and, 1.3 area%, respectively. Their reproducibility standard deviations were 0.33, 0.18, and 0.19, respectively. The mean C4:0, C6:0, and C8:0 contents at the sn-2 position were 0.3, 0.4, and 1.0 area%, respectively. Their reproducibility standard deviations were 0.17, 0.11, and 0.19, respectively. The reproducibility standard deviations of C4:0, C6:0, and C8:0 FAs at the sn-2 position were either the same as or smaller than those for milk fat, although the FA contents at the sn-2 position were smaller than those in the milk fat. Therefore, it was concluded that the CALB method for estimating the regiospecific distribution is applicable to TAGs containing short- and medium-chain FAs. When estimating the short-chain (SC) FA contents in fats and oils by GC, it is better to analyze SCFAs as PEs or butyl esters, and not as methyl esters, in order to prevent loss of SCFAs during the experimental procedure because of their volatility and water solubility. This study also revealed that the stationary phase of the GC capillary column affected the flame ionization detector (FID) response of SCFAs. The theoretical FID correction factor (MWFA / active carbon number / atomic weight of carbon) fitted well with the actual FID responses of C4:0-C12:0 FAs when they were analyzed as PEs using a DB-23 column; however, this was not the case when the GC analysis was performed using wax-type columns.

Enzymatic Analysis of Positional Fatty Acid Distributions in Triacylglycerols by 1(3)-Selective Transesterification with Candida antarctica Lipase B: a Collaborative Study.

The positional distributions of fatty acids (FAs) in fats and oils are principally analyzed by selectively transesterifying the target triacylglycerols (TAGs) at the 1(3) position using Pseudozyma (Candida) antarctica lipase, followed by recovering the resulting 2-monoacylglycerols (MAGs) by chromatography. FA compositions were measured by gas chromatography (GC) after methylating target TAGs and 2-MAGs. The method was collaboratively evaluated by 12 laboratories by analyzing the positional FA distributions in soybean, palm, and sardine oils. The maximum reproducibility relative standard deviations for the major FAs and those at the sn-2 positions of soybean, palm, and sardine oils were 4.41% and 3.92% (18:3n-3), 4.48% and 3.82% (18:0), and 8.93 and 8.24% (14:0), respectively. The values at the sn-2 position were always low. Therefore, these results indicated that the variations were mainly caused by the FA analysis procedure, i.e., the methylation and GC analyses, rather than the enzymatic transesterification and chromatography utilized to prepare 2-MAGs from the target oil.

Unsaturated aldehydes induce CCK secretion via TRPA1 in STC-1 cells.

SCOPE: Cholecystokinin (CCK) producing cells sense luminal contents to regulate the exocrine pancreas, gastric motility, and appetite. Although long-chain fatty acids (FAs, ≥ C12) are well known to stimulate CCK secretion, the CCK-releasing activities of other aliphatic compounds, such as aldehydes (Alds) or alcohols (Alcs), have not been studied. METHODS AND RESULTS: We tested the CCK-releasing activities of various aliphatic compounds with various carbon chain lengths (C3-C13) and degrees of unsaturation in the enteroendocrine cell line STC-1. CCK released from the cell was measured using an ELISA, and intracellular calcium concentration was measured using Fura-2. Mono- and di-unsaturated Alds at 100 µM, but not saturated Alds, induced CCK secretion in STC-1 cells. Alcs and FAs failed to induce CCK secretion, regardless of carbon chain length or degree of unsaturation. Unsaturated Alds increased intracellular calcium concentration, but saturated Alds, Alcs, and FAs did not. Intracellular calcium mobilization and CCK secretion induced by unsaturated Alds was abolished in the absence of extracellular calcium. In addition, the inhibition of the transient receptor potential ankyrin 1 (TRPA1) channel suppressed unsaturated Ald-induced CCK secretion and intracellular calcium mobilization. CONCLUSION: Unsaturated Alds are potent aliphatic stimulants for CCK secretion through the activation of TRPA1.