Progress 12/22/15 to 10/31/20
Outputs
Target Audience:Target audiences are any groups concerned with oxidative degradation of foods and biological materials. The practical aspects of this project clearly target food processing and stabilization in the food industry, but results are also relevant and applicable to the personal care products industry as well as to physiological toxicology and medicine. The fundamental chemistry studied in this project targets academic researchers and students, seeking to elucidate details of these complex reactions and stimulate others to undertake more detailed research in this field, as well as to teach students about the issues that must be coonsidered in stabilizing foods and related products against oxidation and production of toxic products. Changes/Problems:Investigation of potential tocopherol shifts in oxidation pathways was added to the project after prelminary observations in our laboratory showed increased hydroperoxides in the presence of tocopherols and epoxides decreased at the same time. Then another lab studying food oils containing tocopherols did not see epoxide formation until tocopherols were consumed. Since increased attention is not being given to epoxides, we felt it was important to learn more aout their formation conditions. Also, all our alternate pathway research has been conducted on pure lipids while lipids used in foods always have some antioxidant present. Traditionally, antioxidants have been thought to only quench lipid radicals, nothing else. However, research in our lab and a few others is showing that antioxidants exert much more extensive effects on lipid oxidation and other food components. We felt that including invetigations of tocopherol effects on lipid oixdation pathways was clearly warranted and timely. What opportunities for training and professional development has the project provided?This project consistently provides training for graduate students in research and both graduate and undergraduate students in education. During this past year, two masters students and one PhD student were trained in details of lipid oxidation processes and analyses. During the full term of this project, 2 PhD dissertations and 5 MS degrees on topics of this project were completed, training in lipid oxidation analyses was provided to 3 visiting international student scholars, two PhD students in other universities, 2 undergraduates in formal research projects and 7 undergraduates in an experiential learning semester, and one MS student in a graduate internship. Results of this project have been incorporated into undergraduate Principles of Food Science and graduate Food Chemistry Fundamentals and Lipid Chemistry courses. How have the results been disseminated to communities of interest?During the life of this project, results have been disseminated in four detailed and extensive chapters on lipid oxidation mechanisms, analyses, and toxicity of dietary lipid oxidation products; in 5 published research papers; in 19 papers presented at national and international scientific meetings; and in four seminars for industry or other universities. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
During this project period and summary: Alternate pathways of lipid oxidation: Experiments comparing differences in oxidation pathways for linoleic acid and oleic acid methyl esters and triacylglycerols, delayed curing COVID-19 closures, are in progress. Samples are oxidized at 25, 40, and 60 °C to test effects of elevated temperatures on oxidation pathways and product distributions, then are analyzed periodically for non-volatile conjugated dienes, hydroperoxides, epoxides, carbonyls, and alcohols. Results: methyl oleate accumulates hydroperoxides and carbonyl products much more slowly (weeks vs days) and to substantially lower levels than methyl linoleate. Notably, epoxides are present at 2-3 times the hydroperoxide levels and show rapid transformations to other products. This strongly supports additional studies to investigate the toxic potential of monounsaturated oils as well as the possibility of epoxide re-routing by phenolic antioxidants. Major impact: demonstration that multiple competing pathways do exist in lipid oxidation, that hydroperoxides are not the only early products in lipid oxidation, and that production of early epoxides from lipid peroxyl radical additions and secondary epoxides from lipid alkoxide rearrangements are critically important processes in lipid oxidation. Distributions of lipid oxidation products change markedly with temperature and in open vs closed reaction systems, and these changes are missed with standard analyses of single products Our results argue strongly for broad analyses of multiple lipid oxidation products to accurately determine extent of oxidation in any system. Methodologies for analyzing oxidative degradation: A method using Triphenylphosphine to quantitate nanomolar levels of lipid hydroperoxides, originally developed and validated with cumene hydroperoxides, was extended to lipid hydroperoxides. Validated procedures are being used to track effects of lipid structure and tocopherols on oxidation pathways. Reaction conditions for Dinitrophenylhydrazine (DNPH) reaction with lipid carbonyls (aldehydes and ketones) were modified (increased pH and decreased DNPH concentrations) to avoid protonation of dinitrophenylhydrazine to unreactive form and provide full quantitative reaction with lipid carbonyls. This method detects higher levels of carbonyls from lipid oxidation than previously recognized and is being used in all current experiments. To avoid the non-specificity of complexation reactions, we are analyzing lipid alcohols by direct application of reversed phase (pentafluorophenyl column) to track product shifts induced by tocopherols and normal phase (silica column) HPLC with CAD detection in all other studies. Conversion of LOOH, epoxide, and carbonyl analyses from reversed phase to normal phase HPLC for analysis of triacylglycerols (oils) has eliminated the use of extended gradients and made it possible to elute unreacted lipids within a few minutes while polar products are separated on the column. Core products on the glycerol elute early, followed by small polar products. These methods are being applied to analysis of oxidation pathways in triolein and trilinolein. A charged aerosol detector is being applied to detect and quantitate lipids and lipid oxidation products that lack reactive groups or chromophores. Saturated and unsaturated fatty acid methyl esters and corresponding triacylglycerols (oils) give linear and reasonably comparable responses, and lipid epoxides and hydroxylipids are detected without derivatization. Non-volatile aldehydes give strong linear responses but volatile products are lost in aerosolization. Detecting unoxidized fatty acid as well as all non-volatile versions of all major product classes facilitates determinations of oxidation kinetics and total product distributions. Oxygen bomb instrumentation used for accelerated shelf life testing is being tested to understand chemistry occurring in the sample cell, resolve arguments regarding mismatches between methodologies, and determine appropriate conditions for use of the bomb in lipid oxidation analyses. Separations of methyl ester products on metal-free columns in metal-free reversed phase HPLC and in low-metal normal phase HPLC have been developed to detect all lipid oxidation products simultaneously without derivatization. Normal phase HPLC provides baseline separation of lipid hydroperoxide positional and cis-trans isomers and directly reveals changes in product profiles as oxidation progresses. Major impact: providing analyses essential to follow oxidation pathways: 1) methods for basic research in mechanisms and methods suitable for general research and quality control; 2) increased sensitivity (sub-micromolar levels of lipid products) to detect oxidation at very early stages; 3) detection of products from each product class (e.g. hydroperoxides, carbonyls, epoxides, alcohols) accurately and quantitatively; and 4) assays applicable to structurally complex molecules (triacylglycerols) as well as structurally simple lipids (fatty acid methyl esters). With low detection limits, we are able to determine and compare activities of multiple reaction pathways and distributions of products in very early stages of lipid oxidation. Procedures for detection of lipid alcohols (LOH) are particularly important because LOH have not been previously analyzed, so for the first time we are able to evaluate the full roles of LOH as lipid oxidation products and whether antioxidants shift reactive and potentially toxic products such as epoxides and aldehydes to non-reactive, non-toxic alcohols. Developing these assays that cover all the main classes of lipid oxidation products with high sensitivity and accuracy is a major step forward for increasing the accuracy and achievability of lipid oxidation analyses. Using these multiple analyses together make it possible to identify presence of potentially toxic lipid oxidation products in foods and to tailor processing and stabilization approaches to enhance or eliminate specific products for flavor or reactivity. Analysis and prediction of natural antioxidant effectiveness in preventing oxidation in foods. We reacted small phenolic compounds present in most fruits and herbs with whey protein a-lactalbumin to determine whether reaction of phenols with proteins could divert antioxidant effects away from oxidizing lipids. Reaction extent and effects varied with phenol structure and involved non-covalent associations, covalent linkages involving quinone forms, and induced oxidation of proteins without phenol complexation. Phenols reduced concentrations of protein amino, sulfur, and tryptophan groups, the same residues that are also antioxidants against lipid oxidation. Phenols formed adducts with a-lactalbumin and alters protein structure and emulsification and enzymatic digestibility. Major impact: Results show clearly that phenolic antioxidants have effects in food far beyond inhibition of lipid oxidation. The extent of phenol interaction with this protein suggests that proteins may indeed competitively interfere with lipid oxidation inhibition in foods. Results raises questions about whether phenol interactions are protective or damaging in complex systems because the interplay among oxidizing lipids, proteins, and phenolic antioxidants in foods is quite intricate and multidirectional. Each component reacts with the other two but in different ways. The actions of antioxidants in preserving food stability, nutrition, and safety are so important that unraveling the sequence and preferences in these interactions to determine whether lipid-protein-phenol interactions are competitive, synergistic, or parallel and independent should receive high priority in new research
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
Kandrac, M. 2018. Factors affecting the 2,4-dinitrophenylhydrazine reaction with lipid carbonyls, M.S. thesis, Dept of Food Science, Rutgers University, New Brunswick, NJ.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Indumathi Kangampalayam Palaniswamy. 2019. Hydroxy lipids: Significance and analytical methods, M.S. thesis, Dept of Food Science, Rutgers University, New Brunswick, NJ.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Izzo, C. Investigation of lipid oxidation in model solid food systems via Near Infrared Spectroscopy, PhD. dissertation, Dept of Food Science, Rutgers University, New Brunswick, NJ.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Puganen, A., Kallio, H., Schaich, K., Suomela, J-P., and Yang, B. 2018. Antioxidative properties of ethanol extracts of press residues of currants and sea buckthorn berries, J. Agric. Food Chemistry 66 (13):34263434.
- Type:
Book Chapters
Status:
Published
Year Published:
2020
Citation:
Schaich, K.M. 2020. Toxicity of dietary oxidized lipids, In Baileys Industrial Oil and Fat Products 7th Edit., Shahidi, F., ed., John Wiley, New York, 1-72 (on-line version).
- Type:
Book Chapters
Status:
Published
Year Published:
2020
Citation:
Schaich, K.M. 2020. Lipid oxidation: New perspectives on an old reaction, In Baileys Industrial Oil and Fat Products 7th Edit., Shahidi, F., ed., John Wiley, New York, 1-88 (on-line version).
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Target audience is anyone interested in how foods oxidize and degrade, how food properties and qualities change in the process, how the oxidation can be measured, and how it can be minimized or controlled during production, distribution, and storage to extend food shelf life and maintain food safety and nutritional value. Specific groups reached during this project period were the food industry plus academic or government scientists interested in lipids, proteins, or antioxidants through presentations at professional meetings, food companies through consulting and educational seminars, and graduate and undergraduate students through lectures in three different courses. Changes/Problems:Detailed sensitive analyses of multiple lipid oxidation products are required to elucidate oxidation pathways. Methods were not available for all product classes when this work was initiated (previous Hatch project), and many methods in use were old, not sufficiently sensitive, and often inaccurate. This research lab evaluated existing methods to determine best procedures and developed new assays to increase sensitivity and accuracy. At the beginning of the current Hatch project, only hydroxylipid analyses remained to be developed for use in studies of lipid oxidation, thermal degradation, and protein co-oxidation studies. However, limited funding for other areas have halted areas 2, 4, and 5 in the project, and unforeseen events plus the critical need for multiple analyses in mechanism studies have forced a focus on methods for analyzing lipid oxidation products. Recently the xylenol orange assay adopted for hydroperoxide analyses became unavailable, necessitating rush development of the triphenylphosphine assay in its place. At the same time, the oldest HPLC system became unrepairable, so all product assays involving derivatives had to be run on the remaining HPLC system. Under these conditions, multiple assays could not be conducted simultaneously on lipid samples. Reallocation of USDA grant funds to purchase another HPLC was requested in Aug 2018; authorization was not given until November, 2019. With these funds, a normal phase HPLC system will be purchased to facilitate analyses of triglycerides and may be used for multiple assays with fewer changes in solvents and no change in columns. Very recently, a used GC-MS system in excellent condition was donated to this research project. In addition, industry has been expressing interest in analytical methods for the multiple products targeted in this project, and they need simpler analyses of product classes that can be handled readily in quality control labs. Thus, the final year of this project will focus on integrating the non-volatile analyses developed with volatiles analyses by GC to gain new information about alternate pathways of lipid oxidation (areas 1 and 3 in the project proposal) and on developing simpler versions of the assays that can be used in the food industry. What opportunities for training and professional development has the project provided?Provided research opportunities in lipid oxidation mechanisms for one PhD student and one MS student, lipid co-oxidation of proteins in sausage and prevention of both with antioxidants for one MS student, assessment of chlorosulfonic acid reaction as a potential assay for lipid alcohols for one MS student intern. How have the results been disseminated to communities of interest?Results have been disseminated in two book chapters, one completed thesis, lectures in courses, presentations at professional meetings, seminars for companies. What do you plan to do during the next reporting period to accomplish the goals?1) Elucidation of lipid oxidation mechanisms and products. Complete comparisons of oleic and linoleic acids in incubation studies, tracking conjugated dienes, hydroperoxides, epoxides, carbonyls, and hydroxylipids to determine active degradation pathways. Add studies of lipid oxidation in oils (triolein and trilinolein). Determine effects of reaction conditions on product mixes, especially temperature, oxygen, solvent, and presence of antioxidants. Metal interactions will be included if time allows. 3) Improved analyses of lipid and protein oxidation. Hydroxylipid (LOH) assays. It is critical to establish the extent to which LOH is formed during lipid oxidation in order to track alternate pathways and determine effects of antioxidants. Thus, developing and applying useful LOH assays has very high priority. We are now evaluating a) an assay that reacts dimethyglycine with lipid alcohols and detects products directly by mass spectral analyses, b) electrochemical detection during HPLC, and c) direct detection of lipid alcohols in oxidized lipids separated directly on HPLC columns by comparison of retention times to prepared standards. We will use the method that provides most accurate detection in shortest time in the mechanistic studies noted above. Identification and quantification of lipid oxidation products directly by electrochemical detection in HPLC. A metal-free HPLC system equipped with optical, electrochemical, and corona discharge detectors will be applied to separation and identification of total oxidation products in fatty acid methyl esters for mechanism studies. Gold and platinum electrodes will be tested for sensitivity and effectiveness in detecting the various classes of lipid oxidation products. To protect the column, electrolyte will be added via post-column delivery before the detector. After determination of appropriate potentials for each product (hydroperoxides, epoxides, carbonyls, alcohols), the detection mode will be applied directly to samples of neat underivatized lipids to obtain more accurate quantitation of oxidation. Extension of assays to triglycerides. Current HPLC-based assays of hydroperoxides, carbonyls, and epoxides have been optimized for free fatty acids or methyl esters and can run simply on reversed-phase columns. Complex gradients have been developed to extend these analyses to triglycerides, but the resulting analyses are too long for handling multiple samples without changes in oxidation. To overcome this problem, assays will be switched to normal phase columns which will allow rapid elution of all non-oxidized hydrophobic triglycerides while retaining and separating oxidation products. Analysis of volatile lipid oxidation products by GC/MS. A good GC-MS system with direct injection capability was recently donated to this project. Thus, analyses of volatile lipid oxidation products will be run concurrently with assays of non-volatile products in all future studies. Identify lipid radicals by electron paramagnetic resonance (EPR). EPR analyses using spin traps will be added to product assays to distinguish intermediate radicals dominant under different conditions. This should provide information important for clarifying oxidation pathways.
Impacts
What was accomplished under these goals?
Oxidation of polyunsaturated fatty acids is the major chemical reaction that degrades food quality and nutritional value during storage. After elimination of micro-organisms, lipid oxidation is the biggest problem in food stability faced by the food industry. Because of this reaction, current reformulation of foods with higher levels of polyunsaturated fatty acids for health has dramatically increased problems with stability and development of "rancidity" in foods. This produces off-odors and flavors as well as potentially toxic products while at the same time causing loss of edible food supplies. Industry attempts to limit oxidation with antioxidants and packaging, but current strategies are finding limited success or are failing outright due largely to outdated and incomplete understanding about how lipids oxidize and to inaccuracies in determining the extent of lipid oxidation in foods. To address these problems, this project focuses on learning more clearly how lipids oxidize and on developing improved analytical tools for following the progress of lipid oxidation and quantitating it, for determining how oxidized a food really is and what reactive compounds may be present.. Traditional thinking holds that lipids oxidize by a straightforward free radical chain reaction that is driven exclusively by hydrogen abstraction and generates first hydroperoxides, then secondary products that derive only from hydroperoxide decomposition. This program has identified several alternate pathways that compete with hydrogen abstraction and generate additional products in a much more complex process. To study these pathways, this program has developed more sensitive and accurate analyses for the various lipid oxidation products; many of these assays were not available previously. Testing alternate pathways has demonstrated that epoxides are produced in parallel with or even preceding hydroperoxides. This is a critical observation since epoxides are very reactive with proteins and DNA, and they have high potential for reaction with proteins in foods and for potential toxicity. In addition, epoxides are seldom measured, so actual levels of lipid oxidation in oils and foods may be greatly underestimated. We have also learned that antioxidants don't just stop lipid oxidation - they cause shifts in pathways and products! Current practices of measuring only hydroperoxides miss these shifts and often misinterpret antioxidant effects. Lipid alcohols (hydroxylipids) are almost never measured but they are now known to serve as important signaling agents in vivo and in foods they are important indicators of hydroperoxide decomposition and antioxidant action. Aldehydes are the products tasted and smelled most by consumers and they also react strongly with proteins, causing browning and degrading food quality. While studying the complexities of lipid oxidation reactions, this project is developing easy-to-run and accurate analyses for all these additional products, with different methods for basic research and for quality control in the food industry. Results of this program are drawing attention to the complexity of lipid oxidation, stimulating attempts to detect epoxides and other alternate products among basic researchers and encouraging food companies to consider alternate oxidation pathways and products in evaluating food stability. Ultimately, what we learn about lipid oxidation pathways will be applied to reduce oxidation and increase shelf life, long term quality and nutrition, and safety of foods. Accomplishments this year include the following: 1) Elucidation of lipid oxidation mechanisms and products. Progress was delayed due to need for new analyses and lack of critical instrumentation. See section on project change and redirection for details. 3) Improved analyses of lipid oxidation. Major efforts this year focused on refining assays to detect low levels of lipid oxidation products at equal sensitivity. This was necessary to overcome problems in assays used previously or provide assays not yet available. Goals were to develop two levels of assays, one for identifying individual products in basic research on reaction pathways and another to accurately quantitate classes of products in quality control in the food industry. Most assays in common use are limited to millimolar oxidation products, but these levels accumulate when lipid oxidation is already established. This project (and the food industry) needs to detect a broad range of concentrations, from trace levels of products at the beginning of oxidation through high levels in advanced oxidation. Work this year focused on putting in place accurate assays for hydroperoxides, epoxides, aldehydes and ketones, and alcohols (hydroxylipids) produced in fatty acids and oils, sensitive at the micromolar level, and able to handle a large number of samples. The xylenol orange assay for hydroperoxides (detects micromolar levels) we used previously is available commercially in a kit. During the past year, the kit reagents became undependable and it is difficult to run this reaction "from scratch" due to trace metal contamination that interferes. Thus, we shifted efforts to development of an assay using triphenylphosphine (TPP) reduction of hydroperoxides. The assay could not be run in test tubes and monitored optically because the spectra of the TPP and its product overlapped too much. Thus, the procedures were adapted to reaction in a test tube followed by separation and quantitation of reactants and products by HPLC. High purity cumene hydroperoxide was used as a standard to test and optimize the procedures (both reaction and HPLC conditions), then the method was extended and modified for use with oxidized lipids. HPLC offers the advantage of being able to follow the progress of reaction and ensure that all hydroperoxides are reacted, an important consideration when analyzing lipids of unknown history and oxidation level. This assay is now ready to use in full experiments. Major attention was also given to developing assays for hydroxylipids, for which good methods are not currently available for lipids but are desperately needed to accurately follow the progress of lipid oxidation. All methods in use require transformations that not only derivatize the alcohols but also modify original lipids to produce new alcohols. They also are extremely time-consuming and can handle only a few samples simultaneously. To avoid these problems, we focused on derivatizations able to be applied directly to lipids, with reactions run in a test tube and followed optically, and claimed in the literature to be specific for hydroxyl functional groups on alcohols. We tested several options but were alcohol-specific as claimed and all reacted with other oxidation products as well. Of these, chlorosulfonic acid reaction with alcohols appeared to be most promising in preliminary testing. Tests with saturated lipid alcohols gave straightforward reactions with single products. However, reactions with unsaturated alcohols gave multiple products that were difficult to quantitate, and mass spectral analyses proved that the chlorosulfonic acid reacts at some level with all lipid oxidation products. To provide an easier method for quantitating carbonyl oxidation products in quality control, the dinitrophenylhydrazine (DNPH) reaction used to identify individual aldehydes and ketones was modifed by reducing the DNPH-carbonyl complexes with 2-picoline borane. Under these conditions, all products elute together in HPLC so only a single peak needs to be measured. The test has been validated with mictures of model aldeydes and is now being applied to analyses of oxidized lipids.
Publications
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Zachary Yeager. 2019. Development of HPLC-triphenylphosphine assays for lipid hydroperoxides, M.S. thesis, Dept of Food Science, Rutgers University, New Brunswick, NJ.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Target audiences included undergraduate and graduate students, the food and oils industries, food science professionals in the american Oil Chemistrs' Society, the Institute of Food Technology, and NIFA Principla Investigators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training in lipid oxidation pathways and analyses was provided to one undegraduate and five graduate students. In addition, information gained in this project has been used and communicated in research consultations with food companies. How have the results been disseminated to communities of interest?Results have been disseminated in presentations at professional meetings, seminars in the university, lectures in graduate and undergraduate classes, two book chapters, two Masters theses, and one PhD dissertation . A number of journal articles based on results are in preparation. What do you plan to do during the next reporting period to accomplish the goals?1) Elucidation of lipid oxidation mechanisms and products. Complete comparisons of oleic and linoleic acids in incubation studies, tracking conjugated dienes, hydroperoxides, epoxides, carbonyls, and hydroxylipids to determine active degradation pathways. Add hydroxylipid analyses to studies of linoleic acid oxidation pathways. Begin studies of lipid oxidation in triolein and trilinolein. 2) Elucidation of thermal degradation mechanisms of lipids. Complete publication of previous studies. Begin electron paramagnetic resonance (EPR) studies of thermal scission radicals using in situ heating with and without spin traps. 3) Improved analyses of lipid and protein oxidation. Assays of hydroperoxides, carbonyls, and epoxides have been optimized for free fatty acids or esters in model systems. Two adaptations are now needed for tracking lipid oxidation in oils and lipid extracts: adapt procedures for solution analyses of total products without structure differentiation for quality control assays, and determine core products still attached to triacylglycerols (TAGs) by extending HPLC gradients or by releasing fatty acids from TAGs by lipases. Two derivatizations of hydroxylipids have been identified for testing to develop simple optical methods for quantitative class analyses of these products. A metal-free HPLC system has been obtained to eliminate metal decomposition of lipid oxidation products during HPLC analysis. Equipped with optical, electrochemical, and corona discharge detectors, this system will be applied to separation and identification of total oxidation products in fatty acid methyl esters for mechanism studies. 4) Elucidation of protein oxidation mechanisms. No studies planned until new funding is obtained. 5) Investigations of how natural antioxidants act in food systems. Complete detailed analyses of phenol-induced diversion of oxidation pathways in lipids, e.g. from epoxides to hydroperoxides and from scission carbonyls to hydroxylipids. Such shifts can seriously complicate interpretation of lipid oxidation and antioxidant effectiveness base on current analyses of hydroperoxides and volatile secondary products.
Impacts
What was accomplished under these goals?
3) Improved analyses of lipid and protein oxidation. The traditional method for detecting carbonyl compounds (reaction with dinitrophenylhydrazine, DNPH) has presented numerous problems when used for quantitation rather than mere detecting presence. In refining this assay, conditions under which saturated and unsaturated aldehydes reacted to completion within 20 minutes with minimal generation of hydrazone isomers and no carbonyl condensation products were identified as pH 2.52 with a molar ratio of 2.5:1 2,4-DNPH:Carbonyl. Reaction slopes for the various aldehydes varied by <10% in contrast to previous observations of large differences with aldehyde structure. Reaction variability was <2%, and lower limits of detection and quantification were <50 mg/L. Traditional HCl was replaced by formic acid as the acidifying reagent to make this assay compatible with mass spectrometry analysis of product structure. Triphenylphospine (TPP) has been extensively used to detect traces of hydroperoxides in solvents, and nanomolar sensitivity has been claimed. We are investigated whether theTPP reaction could be adapted for quantitative analyses of lipid hydroperoxides. Solution assays are not feasible since TPP and its product TPPO (formed after reaction with hydroperoxides) have optical absorption maxima that are very close, so absorption changes little as reaction progresses. However, conditions have been identified by which TPP, TPPO, and reacting standard hydroperoxides (e.g. cumene and t-butyl) can be continuously monitored and quantified by reversed phase HPLC as reaction time progresses. Protocols required for reproducible solubilization of TPP, complete reaction, and product stability have been identified. Application of the assay to oxidizing lipids found slow reactions, probably due to steric hindrance from the long acyl chains; reaction conditions are being adapted to provide full reaction with lipid hydroperoxides in both free fatty acids/esters and triacylglycerols (oils). Hydroxylipids (LOH), a major product written in the traditional free radical chain reaction, are almost never measured because they do not contribute recognizable off-odors and flavors, and sensitive practical methods of analysis are not available. However, they have important physiological roles and their levels may be paradoxically increased by antioxidants, so their routine measurement is critical for accurately assessing lipid oxidation. Comparisons of existing NMR, IR, GC, and HPLC analyses for hydroxylipids for sensitivity, accuracy, ease of use, and ability to handle large numbers of analyses have been completed. NMR, GC, and HPLC methods were considered to time-consuming for routine analyses and present high likelihood of modifications of lipid oxidation products during sample preparation. As alternatives, two optical methods using derivatization of hydroxyl groups by chlorosulfonic acid/pyridine or by antroyl cyanide are being investigated for sensitivity, accuracy, and ease of use in routine analyses. Chemical analyses of multiple lipid oxidation products are exceedingly tedious to handle and require long times to complete. The food industry needs methods that can be used at-line or for fast turn-around in quality control labs. To provide an instrumental method as an alternative, Near IR (NIR) analyses of lipid oxidation have been tested using standard chemical analyses of lipid oxidation for calibration and application of chemometrics for differentiation and quantitation. The natural non-homogeneity of food materials and their packing in sample vials was found to present serious problems for reproducibility and data scatter. To overcome this, a pressure method for forming tablets with homogeneous packing was developed first. Then to micronize samples, the large petri dishes used for sampling were replaced with small shell vials and these were rotated with offset center to maximize the number of points scanned in the sample. To test what oxidation NIR could detect, samples of canola and walnut oils with different levels of unsaturation were oxidized at 40 deg C for up to 8 weeks. Conjugated dienes, hydroperoxides, and carbonyls were analyzed chemically for comparison with features in NIR spectra. The oils were then mixed with rice flour to provide a solid matrix simulating foods, pressed into pellets, and analyzed with rotation and large numbers of scans to obtain representative pictures of lipid oxidation in a non-homogeneous matrix. Application of chemometric analyses found that NIR spectra correlated strongly with conjugated dienes, which are the first changes that occur in lipids during lipid oxidation. Interestingly, the conjugation arose from a combination of conjugated carbon-carbon double bonds in lipid chains and aldehydes with double bonds at carbon 2. Initial correlations between chemical assays of hydroperoxides and NIR spectral components were too low to be useful in following and predicting lipid oxidation. However, further investigations showed that when the oils were mixed with the rice flour, the lipid hydroperoxides both decomposed to epoxides and carbonyl products and reacted with amine groups in the flour proteins. All of these components were identified in NIR spectra and provided strong correlations with lipid oxidation in the original oil. From these results, guidelines for physical handling of samples in NIR analyses, developing calibrators for quantitation of lipid oxidation products in NIR, and for applying chemometrics to mine data about lipid oxidation from NIR spectra are being developed. 5) Investigations of how natural antioxidants act in food systems. Natural antioxidants often show disappointing effectiveness in stabilizing foods, possibly because phenol reactions are diverted from lipid radical quenching to complexation with proteins. To investigate the extent to which antioxidants may depleted by reaction with proteins, eight polyphenols found commonly in foods or natural extracts were reacted with purified a-lactalbumin (ALA) in solutions buffered at pH 7. Chemical analyses showed that phenols differed in their reactivity with protein amine, thiol, disulfide, and tryptophan groups, with gallic acid, pyrogallol, and hydroquinone being generally most reactive. These three phenolic compounds were highly reactive with sulfur amino acids groups, complexing with up to 90% of cysteine free or from reduced cysteine(-SH groups) in ALA. They also formed non-covalent associations with about 25% of unreduced cystine (S-S bonds) and covalent linkages with small amounts of these bonds, with limitations most likely caused by steric inaccessibility in the proteins. Fluorescence analyses showed that hydroquinone, resorcinol, and catechol bind strongly to three tryptophan residues in hydrophobic environments, while pyrogallol and phenolic acids bind more weakly to a single tryptophan near the protein surface. Since tryptophans are all in the protein interior, such reaction suggests that phenols can induce opening of the protein structure with at least partial denaturation. This possibility was verified by circular dichroism, which revealed that all phenols opened the random coil structure of the protein. Gallic acid and pyrogallol (three phenolic groups on the aromatic ring) and phenolic acids altered protein structure by decreasing a-helices but increasing b-sheet structures, while hydroquinone, catechol, and resorcinol (two phenolic groups) did the opposite (increased a-helices, decreased b-sheets). All the phenolic compounds reacted moderately with free amines in the ALA. Although this reaction was less than expected from previous results, it still provides a means by which proteins are protected and system oxidation is reduced by phenols even while antioxidant protection is diverted away from lipids. This complex action of phenols in foods must be recognized and accounted for in designing antioxidant formulations with natural antioxidants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Saade, C., Gualtieri, A.J., Schaich ,K.M., Yam, K.L., Annous, B.A., and Liu , L-S. 2018. System Feasibility: Designing a Chlorine Dioxide Self-Generating Package Label to Improve Fresh Produce Safety Part II: Solution Casting Approach, J. Innovative Science and Emerging Technology, 47:110-119.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Burgess, B,,Melis, M., Scoular, K., Driver, M., Schaich, K.M., Keller, K.L., Barbarossa, I.T., and Tepper, B.J. 2018. Effects of CD36 Genotype on oral perception of oleic acid supplemented safflower oil emulsions in two ethnic groups: A preliminary study, J. Food Science, 83(5): 1373-1380.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:The target audience is any group interested in or affected by lipid oxidation and needing to counteract lipid oxidation in any material. The immediate focus of this project is obviously mostly on the food industry. However, lipid oxidation is broadly involved in many physiological and toxicological cell processes in both plants and animals, so the new information about lipid oxidation pathways will also be critical for biochemical, toxicological, and medical research. Since oils are important functional ingredients in many health care products, including soaps and lotions, the cosmetics and health care industries are another target sub-group. Finally, students learning about lipids and lipid oxidation are a key target audience for learning and education in both basic and applied areas. This includes undergraduate and graduate students in food science, chemistry, biochemistry, physiology, toxicology, and medicine. Information was delivered to these target audiences through classroom teaching, student research projects, industrial short courses and individual presentations, consulting, and scientific publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Research training was provided for six graduate students and seven undergaduate students. How have the results been disseminated to communities of interest?Results of these studies have been presented in five oral papers at professional meetings and in seminars at two universities and one international company, they are being incorporated into three courses taught by the PI at Rutgers, and they are being written into several chapters for books. The importance of alternate pathways in accurately analyzing lipid oxidation and identifying toxic potential has been discussed with two companies, and collaborative research projects are being developed. What do you plan to do during the next reporting period to accomplish the goals?1) Elucidation of lipid oxidation mechanisms and products. Add oleic acid to incubation studies, tracking conjugated dienes, hydroperoxides, epoxides, carbonyls, and hydroxylipids to determine active degradation pathways. Add hydroxylipid analyses to studies of linoleic acid oxidation pathways. Begin studies of lipid oxidation on inert solid surfaces to determine effects of matrix on oxidation pathways. 2) Elucidation of thermal degradation mechanisms of lipids. Begin electron paramagnetic resonance (EPR) studies of thermal scission radicals using in situ heating with and without spin traps. 3) Improved analyses of lipid and protein oxidation. Complete testing of TPP assay for hydroperoxides and determine whether it can be used in subsequent lipid oxidation studies. Complete DETC solution assay for epoxides and test with standards and oxidized oils. Develop solvent gradient to extend HPLC analysis of DETC complexes to triglycerides and oils. Test HPLC and GC methods for hydroxyl lipid analysis; determine best approach for use in mechanisms studies. Begin spin trap studies to determine specific lipid radicals with electron paramagnetic resonance (EPR). 4) Elucidation of protein oxidation mechanisms. No studies planned until new funding is obtained. 5) Investigations of how natural antioxidants act in food systems. Complete studies of lactalbumin reactions with phenolic compounds; confirm results in a new protein. Begin detailed analyses of phenol-induced diversion of oxidation pathways from epoxides to hydroperoxides and from scission carbonyls to hydroxylipids.
Impacts
What was accomplished under these goals?
Impact Lipid oxidation is the major chemical reaction limiting shelf life of foods. Recognition of the important role of polyunsaturated fatty acids in health has stimulated reformulation of foods with higher levels of these lipids. However, doing this has dramatically increased problems with stability and development of "rancidity" which produces off-odors and flavors and potentially toxic products while causing loss of food supplies. Traditional thinking holds that lipids oxidize by a straightforward free radical chain reaction that is driven exclusively by hydrogen abstraction and generates first hydroperoxides, then secondary products that derive only from hydroperoxide decomposition. Stabilization using approaches based on this reaction sequence has had only limited success, or even failure. This program has identified several alternate pathways that compete with hydrogen abstraction and generate additional products in a much more complex process. To study these pathways, this program has focused on development of more sensitive and accurate analyses for the various products, many of which were not available previously. Testing these pathways has demonstrated that epoxides are produced in parallel with or even preceding hydroperoxides. This is a critical observation since epoxides are very reactive with proteins and DNA, and they have high potential toxicity. In addition, they are seldom measured, which means that actual levels of lipid oxidation in oils and foods may be greatly underestimated. We have also learned that antioxidants don't just stop lipid oxidation - they cause shifts in pathways and products. Current practices of measuring only hydroperoxides miss these shifts and often misinterpret antioxidant effects. Overall, this program is drawing attention to the complexity of lipid oxidation, encouraging food companies to consider alternate oxidation pathways and products in evaluating food stability and developing sensitive and accurate analytical methods to provide tools needed for analyses of lipid oxidation. Outcomes 1) Elucidation of lipid oxidation mechanisms and products. A grant proposal for new research in this area submitted to USDA NIFA was funded. 2) Elucidation of thermal degradation mechanisms of lipids. Manuscripts for journal articles from four theses or dissertations in this area are being written 3) Improved analyses of lipid and protein oxidation. The traditional method for detecting carbonyl compounds (reaction with dinitrophenylhydrazine, DNPH) has presented numerous problems and inconsistencies when used for quantitation rather than mere detecting presence. Hence, the detailed chemistry of the reaction was re-evaluated and conditions required for full reactivity without side reactions were investigated. Excess acid was found to be a problem with previous protocols. A pH of 2.2-2.5 was found to be optimum for forming the intermediate carbocation on the carbonyls while avoiding competing isomerization and carbonyl condensation. Conditions for ensuring complete reaction of all carbonyls have been identified, and HPLC separations have been improved to reduce overlap of critical pairs (unsaturated aldehyde and saturated aldehyde two carbons shorter). Triphenylphospine (TPP) has been extensively used to detect traces of hydroperoxides in solvents and nanomolar sensitivity has been claimed. We are investigating whether theTPP reaction could be adapted for quantitative analyses of lipid hydroperoxides. Solution assays are not feasible since TPP and its product TPPO (formed after reaction with hydroperoxides) have optical absorption maxima that are very close, so absorption changes little as reaction progresses. Thus, we are seeking to track the reaction using reversed phase HPLC to simultaneously monitor TPP, TPPO, and reacting standard hydroperoxides (cumene and t-butyl) as reaction time progresses. Results to date reveal problems with the stability of both TPP and TPPO, and show that the stoichiometry of the reaction (i.e. how much TPP is consumed and how much TPPO is generated per mol of hydroperoxide) is inconsistent. Patterns suggest that unidentified side reactions also consume TPP and dissipate TPPO. If these side reactions cannot be identified and eliminated, TPP may not be a feasible agent for quantitating lipid hydroperoxides. Hydroxylipids (LOH) are a major product written in the traditional free radical chain reaction, yet are almost never measured because they do not contribute recognizable off-odors and flavors, and sensitive practical methods of analysis are not available. However, hydroxylipids have important physiological roles and their levels may be paradoxically increased by antioxidants, so their routine measurement is critical for accurately assessing lipid oxidation. Existing NMR, IR, GC, and HPLC analyses for hydroxylipids have been evaluated and compared for sensitivity, accuracy, ease of use, and ability to handle large numbers of analyses, e.g. in shelf life studies. NMR is simplest in handling and can detect multiple oxidation products in the same sample, but detects only 0.1 mM products at most. All the GC methods require extensive sample modification and clean-up and are not amenable to multiple sample testing. We will start testing HPLC methods in the coming year. Previous analyses of epoxides in this program quantitated epoxides by complexation with diethyldithiocarbamate, DETC, followed by separation of products by HPLC. This works well when purified fatty acids are being oxidized but oils and extracts will require application of complex gradients. Since industry needs a protocol that is fast, easy, and accurate for quantitation of total epoxides without differentiation of structure, we have attempted to convert the DETC reaction to a test tube assay that will detect all epoxides, including those on triacylglycerols, in a one-step optical assay. As with the triphenylphosphine assay for hydroperoxides, the starting material and complex have absorption maxima that are close together. However, in this case, phosphoric acid can be added to decompose unreacted DETC, thus revealing the complex. Results have been promising and epoxides have been detected in oil samples using preliminary protocols. HPLC is being used to follow the reaction and track reaction kinetics and products for verification that the reaction is complete and stoichiometric. This data will be used to validate the optical method.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Schaich, K.M. 2017. Thinking outside the classical chain reaction box of lipid oxidation.. Evidence for alternate pathways and the importance of epoxides, Lipid Technology, 29(9-10): 91-96.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Saade, C., Annous, B.A., Gualtieri, A.J., Schaich ,K.M., Liu , L-S., and Yam, K.L. 2017. System Feasibility: Designing a Chlorine Dioxide Self-Generating Package Label to Improve Fresh Produce Safety Part I: Extrusion Approach, J. Innovative Science and Emerging Technology, 43(10): 102-111.
- Type:
Book Chapters
Status:
Published
Year Published:
2017
Citation:
Schaich, K.M. 2017. New Perspectives in Lipid Oxidation, In: Food Lipids-- Chemistry, Nutrition, and Biotechnology, Fourth Edition, C. Akoh, Ed., CRC Press, Boca Raton, FL, pp 481-499.
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Progress 12/22/15 to 09/30/16
Outputs
Target Audience:The target audience is any group interested in or affected by lipid oxidation and needing to analyze lipid oxidation products in any material. The immediate focus of this project is obviously mostly on the food industry. However, lipid oxidation is broadly involved in many physiological and toxicological cell processes in both plants and animals, so the new analytical methods refined during this reporting period will be criticalalso for biochemical, toxicological, and medical research. Since oils are important functional ingredients in many health care products, including soaps and lotions, the cosmetics and health care industries are another target sub-group. Finally, students learning about lipids and lipid oxidation are a key target audience for learning and education in both basic and applied areas. Information was delivered to these target audiences through classroom teaching, student research projects, industrial short courses and individual presentations, and consulting, and scientific publications. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The DNPH carbonyl analysis provided technical and research training for one graduate student and forms the basis of a MS thesis which will be defended in early 2017. The DETC epoxide analysis provided technical and research experience for an undergraduate student during the summer. THe FT-NIR analyses provided technical and research training for one graduate student and form the basis of a MS thesis which will be defended in early 2017. How have the results been disseminated to communities of interest?Results of this period have not yet been disseminated formally to the scientific community. Two MS theses are in writing stages and should be defended in Jan 2017. After that, the two analytical methods being studied (DNPH analysis of lipid carbonyls and FT-NIR analyses of oxidized lipids) will be published. Fundamental aspects of these two analyses were discussed in the chapter on lipid oxidation analyses cited above and have been shared with colleagues at meetings.. What do you plan to do during the next reporting period to accomplish the goals?From the accomplishments section above, we will continue our work and analysis on the subprojects we have begun.
Impacts
What was accomplished under these goals?
This project focuses on elucidating reactions involved in oxidation of foods in order to improve stabilization and protect food nutrition and sensory quality. A major requirement to accomplish this is developing laboratory protocols for detecting and quantitating oxidation products. Oxidation is the major chemical reaction leading to degradation of foods, after spoilage by micro-organisms has been eliminated. In what is known commonly as "rancidity", unsaturated lipids oxidize first, generating free radicals and a variety of products, including epoxides, aldehydes, ketones, and polymers. Some of these products are potentially toxic so it is critical to begin documenting their production in oils and foods. Oxidizing lipids then react with other molecules in foods, including proteins, starches, vitamins, pigments, and DNA to cause damage that diminishes nutritional value, changes food structures, generates off-flavors and odors, and removes natural flavors. To learn about the chemistry underlying these changes, we need to be able to analyze both the lipid oxidation products reacting with other molecules and also the damage caused. As this period was the start of a new Hatch project and is in the rebuilding stage, we focused on refining and validating three important assays: chemical assays for non-volatile lipid carbonyl and epoxide products that included both solution assays for total class quantification and HPLC analyses to determine individual products, and instrumental FT-NIR (Fourier transfer near infra-red) analyses that detect functional groups on molecules in intact food products. The chemical analyses are for use in research and industrial quality control; the FT-NIR analysis will be useful in basic research but will be very important for monitoring oxidation on-line in food production and storage facilities. Analysis of lipid carbonyl products by reaction with dinitrophenylhydrazine (DNPH) and separation of products by high pressure liquid chromatography (HPLC). Formation of hydrazones from DNPH is the classical reaction for detecting carbonyl compounds but application of this assay to oxidized lipids has not given consistent, reproducible, or quantitative results. Since alternative carbonyl assays are not available, this project has sought to debug this assay and determine conditions for optimal and valid use. The assay itself is very simple but the reaction of DNPH is rather complicated. Excess acid appears to be a common condition for studies reported in the literature. Some acid is required as a catalyst, but too much protonates the DNPH, making it unreactive, and too little allows condensation of the carbonyls before they react with DNPH. In addition, sulfuric acid is most frequently used but it is hard on HPLC columns and cannot be used if mass spectrometry detection is coupled to HPLC. To solve these problems, we investigated effects of acid type and concentration on the efficiency of DNPH reaction with carbonyl standards, following formation of hydrazones and side products over time by HPLC. In the process we learned that the DNPH reaction with short chain saturated aldehydes is fast while reaction with longer chain unsaturated aldehydes (as are common in oxidizing lipids) is slow, differing by hours. Results have been integrated into an optimized assay that is currently being tested with oxidized fatty acids and oils. Analysis of epoxides by reaction with diethyldithiocarbamate, with solution assays for total quantification and HPLC separation of individual products for determinations of reaction mechanism. An HPLC version of this assay had been previous developed in our program, but was time-consuming and only semi-quantitative. Optical analysis of DETC-epoxide complexes was investigated to provide an alternative rapid assay that would detect and quantitate all epoxides in a sample (free and attached to oils or phospholipids). Reaction times and temperatures, epoxide concentration ranges, overlap of DETC and its epoxide products, and acid require for removal of unreacted DETC were all investigated. Completeness of the reaction was then verified by HPLC analyses of the reaction mixtures. An optimized version of the reaction is currently being tested in oils oxidized for the FT-NIR study and extracted from aged dry pet foods. Analysis of lipid oxidation intact foods by FT-NIR. FT-NIR is being investigated as a rapid instrumental assay that may supplement chemical assay in research and replace chemical assays in industry quality control and on-line monitoring. Although FT-NIR use is increasing, the standardization required for the method is seldom done (or done too minimally) so the spectra show that changes occur in oxidized materials but these changes cannot be identified. To provide a sound bases for generating reproducible NIR data and interpreting the results chemically, we modified the instrumentation to accommodate and rotate small samples (grams) and built a press to reproducibly press food materials into pellets for NIR analysis. These procedures reduce the sample variability that has hampered use of this method previously We oxidized oils with three levels of unsaturation, and analyzed their oxidation by conjugated dienes, hydroperoxides, and non-volatile carbonyls. The oils were then mixed with rice flour as a solid support, and analyzed by FT-NIR. Levels of detected products were integrated with NIR spectral characteristics directly and in chemometric analyses. Computer models are being developed to correlate specific lipid oxidation products with specific spectral features and assess how best to apply NIR data in both research and industrial applications. A research proposal "Revised Understanding of Lipid Oxidation Mechanisms" that addresses Objective 2 plans of this Hatch project was submitted to the Foundational Program of NIFA. Notice of approval and funding has been received.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2016
Citation:
Schaich, K.M. 2016. Analysis of lipid and protein oxidation in fats, oils, and foods, In: Oxidative Stability and Shelf Life Of Oils/Fats and Oils/Fats Containing Foods, Hu, M. and Jacobsen, C, Eds., AOCS Press, Champaign, IL. , pp. 1-131.
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