Fish Oil Oxidation and Oxygen Discussion
Dr. Jerome J. King, Dr. Randolph M. Howes, and Dr. Jie Zhang
Marine oils have long been recognized as having health benefits and that mind set is still true today. Modern consumers, in addition to recognizing health benefits, are also concerned about the overall safety of the oils both for themselves and for their loved ones. How does one know if oil is “safe” and does it contain chemicals or chemical breakdown products that may be detrimental? Modern day analytical methods and instrumentation can determine the type and amount of fatty acids in the oil and medical science has established certain health benefits with select fatty acids.
From a strictly biochemical aspect, fats are composed of fatty acids and glycerol. Fatty acids are long-chained organic molecules that can be classified as saturated, monounsaturated, or polyunsaturated. Saturated fatty acids are composed of carbon atoms bonded to the next carbon atom all with single bonds. A mono-unsaturated fatty acid is also composed of carbon atoms bonded together, but two of the carbon atoms have a double bond which is denoted as - C = C -. A polyunsaturated fatty acid has two or more sets of carbon atoms with double bonds and could be denoted as - C = C – C = C -. The number and location of the double bonds affect the physical composition of the fat. If fatty acids with multiple double bonds are present, the fatty acids result in liquid oil while if the fatty acids are all saturated, the material would be a solid. Plant or vegetable oils contain monounsaturated or polyunsaturated fatty acids and are liquid in nature while animal fats, which generally contain unsaturated fatty acids, are solid.
The presence of double bonded (unsaturated) fatty acids also introduces a fatty acid that is more prone to certain chemical reactions (oxidations) from a variety of sources. The reactions can cause breakage of the double bond and the formation of chemicals that cause off-odors and off-flavors to the oil and concerns about the health effects of the product. Fish oil oxidation includes a series of chemical reactions. The oxidation of oil is related to many factors, such as oxygen, temperature, light, moisture and catalysts. The oxidation of oil cannot be stopped completely; it happens in every step of oil production and consumption.
If one or more of the fatty acids is removed from the triglyceride, the fatty acid is called a free fatty acid. Free fatty acids can be produced by enzymatic activity (lipases), oxidation and other chemical reactions. Actually, in the body, most of the fat is hydrolyzed by enzymatic activity to release free fatty acids and mono- or di-glycerides in order for the digestive tract to absorb the fat into the body. Once absorbed, these components will reform triglycerides and involve into metabolism. The fatty acids (including free fatty acids) are important in the metabolism of energy production, energy storage, membrane formation, and cellular signaling transpiration. In industry, some companies use the level of free fatty acids for evaluation of the sales value. The level of free fatty acids can be used as an index for oxidation level in the oil. Oxidation processes can release the free fatty acid from the triglyceride which can happen during prolonged heating at high temperature. The presence of free fatty acids, in turn, can speed up oxidative reactions. There are no official standards for free fatty acids, and again, free fatty acid levels indicate nothing about the health safety by the oil industry. The test for free fatty acids is a titration where any chemical that can neutralize a base is listed as a free fatty acid.
Dave’s comment: Our body metabolizes fats through oxidiation process into free fatty acid form. FFA is a important fat structure our bodies commonly require a fatty acid for digestive ability. The beauty of fermentation is predigesting the raw material through a natural enzymatic/bacterial digestion producing a product that is readily used by our bodies. Our focus is to create FFA for easy digestion of the oil. I suspect one of the reasons that fermented cod liver oil digests easier for most people compared to industrial produced oils is because of the natural FFA in the product. Industry uses a variety of methods to remove FFA from their oils then adds preservatives (anti-oxidants) to hold the product from breakdown/digesting.
Oil oxidation can produce different compounds in different oxidative stages. It starts with an oxidation stage of peroxides and dienes, from which free fatty acids will be produced. Then, the oxidation enters the secondary oxidation stage, where the carbonyls and aldehydes may be produced. The oxidation products may bring an undesirable smell and/or color in the oil, which is the reason the industry would like to use many different processes to remove or prevent the production of oxidation products.
Dave’s comment: I suspect substantial difference in product character based on production practice, fermentation, wild fermentation, or heat/chemical breakdown. With the exception of food safety standards, each end product will have a unique set of standards, markers and expectations.
There are many methods to determine the stages of oxidation. One widely used method is called Peroxide Value (PV), which is used to measure the primary oxidation products. The hydroperoxides, which are the main products from primary oxidation reactions, are measured by the PV test. Chemically, a hydroperoxide is an organic molecule that has carbon groups bonded with two oxygen atoms and one terminal hydrogen and is a derivative of hydrogen peroxide. A high PV indicates that high levels of primary oxidation products are present. The peroxide value test can initially start at a low value, increase over time as primary oxidation occurs, and then drop as the primary oxidation products are reacted upon. The high PV value always relates to high temperature process and long heating time, such as the long-time used oil in the deep fry.
When the hydroperoxides decompose to other compounds, oxidation enters a secondary stage. The secondary oxidation products are measured with the p-Anisidine test, which uses a reagent called Anisidine. When added to samples of oil, the Anisidine reacts with ketones and aldehydes and produces a measurable chemical complex. The test gives an indication of the number/amount of aldehydes and ketones in the product. The higher Anisidine value, the more aldehydes and or ketones present, which can give the oil a more noticeable off taste or odor.
Dave’s comment: I have never seen relevant issues of hydroperoxide formation in any raw fat. I suspect relevant hydroperoxide formation maybe be near impossible if not impossible without heat and other industrial actions.
Some researchers have incorporated the peroxide value and p-Anisidine value into a Totox value. Over time, the peroxide value will decrease because the double bonded carbons and the formation of primary oxidation products will be used up (chemically converted) with the formation of the secondary oxidation products. The secondary oxidation products will remain in the oil and not decrease in concentration, and may result in a rancid oil. To understand oil and its potential of rancidity, it is important to monitor the formation of primary and secondary oxidation products over time. The Totox value equals 2PV + AV, which is used to represent the overall oxidation state. A typical oxidation state can be shown as in the figure below:
Currently, there are no approved international standards for acceptable peroxide values, p-Anisidine values, or Totox values. There are only suggested standards. This is likely due to the formation rate. The type and amount of secondary oxidation products cannot always be predicted and there is a natural variability of unsaturated fatty acids in fish oil. Also, the determination of rancidity is based on a person’s sense of smell or taste and there is no standard used to define off taste or odor.
Dave’s comment: Every product has its own unique thumbprint. Part of the thumbprint can be PV, AV and Totox markers. These are not food safety markers but rather tell the story of a product through time.
Generally, individual companies will set their own acceptable peroxide value, p-Anisidine or Totox value. Of the three, the p-Anisidine and Totox values may have the most significance for measuring oxidation level of the product, which relates to off-odor and color, because of the variable nature of the peroxide value test.
There is another test called the Thiobarbituric acid (TBA) testing to measure the aldehyde products. However, the TBA test can be inaccurate due other components, such as sugars, DNA, etc. Actually, the TBA test is more frequently used in the test for the freshness of animal tissues, such as meat and seafood.
Dave’s comment: TBA is not a marker to be used with oils. It has no bearing or reliable purpose for use in viewing an oils thumbprint.
Because of the potential oxidation of fish oil (due to multiple double bonds in the fatty acids) the levels of unsaturated fatty acids will decrease, as the double bonds are lost. The rate and amount of decrease is variable.
Unlike fat-soluble chemicals such as PCB (polychlorinated biphenyls) and dioxins, the products of lipid oxidation are not considered to be toxic from a chemical point of view. The research on the oxidation by-products indicate isoprostanes and neuroprostanes are produced and neither are found on the California Prop 65 or the Codex Alimentarius.
Prop 65 and Codex are two commonly referenced publications that list toxic or health-related chemicals. The off odors and flavors can be sensory offensive, but chemically, they are considered to be safe and not harmful. The international organizations that set standards for fish oils list requirements for heavy metals such as mercury and organic chemicals such as PCB, but they do not require monitoring of individual lipid oxidation products.
Related to the safety issue for all these oxidation products, Dr. Howes helped us to answer some questions with his expertise.
The following paragraphs were quoted from Dr. Howes’ article.
There is no simplistic solution to the redox dilemmas with which we are faced.
As redox expert, Dr. Barry Halliwell, said, "There is peroxide in a freshly brewed cup of coffee because of the presence of antioxidant polyphenols in the coffee, which serve as electron donors to oxygen." Thus, the more antioxidants present in the foodstuff ingested, the more likelihood for the generation of more reactive oxygen species (ROS), more accurately termed electronically modified oxygen derivatives, (EMODs).
Thus, the addition of antioxidants to fermented food products could serve to create more ROS, not less, if one subscribes to this viewpoint. Ingestion of naturally fermented oxidized products would be expected to generate or produce lower levels of so-called harmful ROS.
Still, the basic premise of ROS being harmful is flawed and outdated but investigators continue to try to force the data to fit their flawed thinking and to support the nullified free radical theory.
It is important to understand that we are not test tubes. While it is true that ROS will attack DNA and proteins, living cells are able to “fight back” against all sorts of stress through special “repair and neutralize” tools. In the case of ROS, there are multiple protection systems: special enzymes like superoxide dismutase and catalase exist for the sole purpose of ROS neutralization, and a small peptide known as glutathione can efficiently scavenge ROS throughout the cell. Despite the existence of these systems, a paradigm has been in place for some years throughout the scientific community that some ROS escape and are able to cause damage to cells, ultimately being responsible for various pathologies and possibly the process of aging. Lately, however, evidence has surfaced that ROS are not simply unfortunate by-products of our aerobic metabolism. ROS are now regarded as important signaling molecules inside the cell, crucial for the communication between different cell compartments. Even more strangely, studies in model organisms have shown that ROS can be media-tors of some health-promoting effects. The most recent of such studies suggests that ROS may be crucial for the proper functioning of the immune system.
Dr. Howes' book entitled, Antioxidants Linked To Deadly Unintended Consequences, presents over 500 study reports showing the ineffectiveness of common antioxidants and of these, 170 study reports show the harmful potential of ingestion of excessive amounts of antioxidants. Because the free radical theory lacks predictability or reproducibility, it has been invalidated and nullified by not meeting the requirements of the scientific method.
Oxidized fat and lipid oxidation products are commonly present in human foods and these compounds are absorbed by the intestine and appear in the blood circulation. These ingested substances may or may not have deleterious effects in both humans and experimental animals.
To our knowledge, there have been no large scale, randomized controlled human trials on these substances. Therefore, considerable additional research is required to establish the extent to which dietary fat oxidation may pose a threat to human health and/or longevity.
Dave’s comment: It is becoming more and more common knowledge that oxygen is healthful rather than harmful in natural metabolic processes. If so, what is in dietary fats that creates damaging issues in our bodies if it is not oxygen. One possible theory goes back to the discussions of Dr. Francis Pottenger, ‘Pottengers Cats’ and Dr. Price’s, ‘Nutrition and Physical Degeneration’. Maybe the problems can be traced to fat production; traditional non-processed fats/raw fats vs. industrial prepared fats. What might cause issues within processed fats? It does not appear to be oxygen. Processed fats are heavily preserved (anti-oxidants) to the point that many processed fats oxidative curves are flat even after a very long exposure to air. Maybe the damage comes from the preservative actions and compounds applied to the processed oils? Or maybe it is one or more of the thousands of altered structures within a processed oil and/or a highly heated oil. I read a study that discussed monounsaturated, polyunsaturated and saturated fats can equally have ill effect on the body. But what I do not recall is a conversation differentiating between the different processing methods to produce the different oils such and raw vs. processed fats.
Hypoperoxides and related oxidative compounds are not relevant in fermented cod liver oil and I suspect will not be relevant in any raw fat.
Ongoing Oxidative Stability Test
We have ongoing oxidative stability testing to compare fermented cod liver oil and two other brand fish oils (brand X and Y). The results are shown below:
There are no official/approved standard measurement values for peroxide value. Some researchers reported that people can smell the off-odor when PV is higher than 20. From the graph, all FCLO results were lower than 20. Compared to the other two brands, brand X had similar results, but there was a sharp peak of brand Y, which appeared after one-week storage.
Dave’s comment: Both brands X and Y use preservatives (anti-oxidants). My guess is brand X is more heavily processed and preserved compared to brand Y. I base this guess on how flat the curve X is when exposed to air. The beauty of fermentation is stability. Refrigeration is a rather new invention. Fermenting foods has been the historically/traditional method to prepare and store foods. It is time tested and proven to work both functionally, safety and best for human health.
The long-term data on PV results can be found in the graph below; all PV results were lower than 10 during 12 month storage period.
Dave’s comment: We have not done extensive studies in this area as these types of tests are not part of food safety discussion but rather for information and study on our fermentation methods. We will learn more over the next couple years.
The results of anisidine value can be found on the graph above. Some commercial oil companies set 20 as a quality control limit for this value. It is clear on this graph that FCLO had the lowest anisidine value. CLO Brand X was higher than FCLO, but CLO Brand Y had a higher number throughout the storage period.
Dave’s comment: None of these results should be used to judge cod liver oils for safety. Over the years we have tested many cod liver oils and to date we have never seen ANY cod liver oil that does not fully meet food safety standards! You can feel safe buying any cod liver oil in the market with full confidence when it comes to food safety concerns!
TBA value was showed above. All CLOs had low numbers of TBA, and there was not a significant difference between the three products. Some references set 5 as a TBA value standard, but this should not become a concern for fish oil products.
Again, all test results showed the FCLO has low oxidation products, which means the oxidation level in FCLO is relative low. Industry has set up oxidation indexes to eliminate off-odor and obtain clearer color.
But, all these oxidation products are not related to any food safety or health issue. The natural fermentation, without heating and light, gives it a unique quality and distinctive health benefits. Therefore, oxidation should not be a concern for this exceptional and great product.
Dave’s comment: Industry does not understand nor respect natural processes. Attempting to define fermented cod liver oil within a industrial context the result will never be flattering as industry standards do not mesh with natural processes. It is like a square peg being pounded into a round hole.
Bio For Dr. Jerome J. King
Currently, Dr. King servers as the Technical Director at Midwest Laboratories, Omaha, Nebraska. Dr. King has been with Midwest since 1989 and has served as the QA/QC Director as well as Technical Director. In his role as the Technical Director and QA/QC Director, Dr. King has provided the oversight for the method development and analytical compliance and certification for a variety of tests, including vitamins, amino acids, cholesterol, and fatty acid profiles. The laboratory has undergone external auditors for ISO 17025 accreditation and has participated in a number of proficiency programs. In the role of QA/QC director, Dr. King has managed the development and implementation of Standard Operating Procedures, internal audits, and the managed the training of new analysts.
Bio for Dr. Howes
Professor Randolph M. Howes M.D., Ph.D. was the first in the history of Tulane School of Medicine to receive double doctorate degrees in medicine and biochemistry simultaneously. He was awarded a patent certificate for inventing the triple lumen venous catheter in 1977, licensed it to Arrow International, Inc. in 1981, successfully defended it is a multimillion dollar six year patent infringement suit and watched it become recognized as the number one venous catheter in the world. His multilumen catheter has been credited with helping save the lives of over 20 million critically ill patients worldwide and the name of Howes is well known in over 100 countries. He retired from his private practice to pursue his dream of contributing to a better understanding of oxygen biochemistry and of conducting an arduous in depth review of the world’s scientific literature on oxygen metabolism. He acquired board eligibility in both general and plastic surgery from training at the prestigious Johns Hopkins Hospital.
He received the 1994 Dr. Norman Vincent Peale Unsung Hero award winner, for his remarkable versatility. Prof. Howes received the Harper Award for innovative research from the American College for Advancement in Medicine and served as their keynote speaker. He is a medical columnist and has written over 500 articles on popular medical topics. In 2013, he was the first doctor in the history of the American College for Advancement in Medicine (ACAM) to be awarded the Charles Farr award for "excellence in oxidative medicine."
In 2004, he published his first in a series of e-books on oxygen metabolism, which was a 767-page tome entitled, “U.T.O.P.I.A.: Unified Theory of Oxygen Participation In Aerobiosis.” In the past eleven years, he published more than 25 books regarding the free radical theory and oxygen free radical metabolism, such as “The Medical and Scientific Significance of Oxygen Free Radical Metabolism.”
Some of Dr. Howes' books are available on www.amazon.com as follows:
[Death In Small Doses? Trafford Publishing, © 2010] [Antioxidant Overkill. © 2011] [Dangers of Excessive Antioxidants in Cancer Patients. © 2011] [Heart Disease and Antioxidant Failures. © 2011] [Antioxidant Failures and Dangers. © 2011] [Anti-Aging Anti-oxidant Scams. © 2011] [Sports, Athletes, Exercise Facts and Antioxidant Myths.
© 2011] [Alzheimer’s Disease: Forget Antioxidants and Supplements. © 2012] [Sex, Performance, Reproduction, Naked Radicals And Antioxidants. © 2012] [Antioxidants Linked To Deadly Unintended Consequences. © 2013] [U.T.O.P.I.A.: Unified Theory of Oxygen Participation In Aerobiosis. © 2014, revised] [Hydrogen Peroxide: A Health, Homeostatic and Protective Essentiality. © 2014] [Reactive Oxygen Species vs. Antioxidants: The Oxypocalypse or The War That Never Was. © 2014] [Diabetes and Oxygen Free Radical Sophistry. © 2014, revised] [FISH OIL [Omega3 fatty acids): Facts, Fantasies & Failures. © 2014] [Vitamin D: Benefits & False claims. © 2014] [Chocolate & Red Wine Antioxidants (Polyphenols, Flavonoids & Resveratrol): Facts vs. Falsehoods. © 2015] [Blueberry, Tomato & CoQ10 Antioxidants (Anthocyanins, Lycopene & Ubiquinone) Claims vs Facts. © 2015]
These books contain over 8,000 pages of material and tens of thousands of peer-reviewed references, which represents the most comprehensive selective overview of oxygen metabolism available today. His belief is that the free radical theory is unfounded and that electronically modified oxygen derivatives (EMODs) are of low toxicity and essential for energy production, for pathogen protection, as secondary cell messengers and as tumoricidal agents. His Unified Theory states that EMOD insufficiency levels “allow” for the manifestation of diseases, including neoplasia and is a contributing factor in the aging phenomenon. He also postulates that an EMOD insufficiency is the basis for coexistence of diseases. Dr. Howes, who is both an experimentalist and a theoretician, is an international lecturer on plastic surgery and a world expert on the biochemistry of oxygen free radicals. His passionate goal is to have cures at the bedside, based on his innovative theories involving electronically modified oxygen derivatives, within his lifetime.
Bio for Dr. Jie Zhang
Dr. Jie Zhang graduated from Sichuan University in Chengdu, China, with a Bachelor of Science degree in Food Science and Technology in June 2006. He earned his Master of Science in Food Engineering from Sichuan University, Chengdu, China, in June 2009. He received his degree in Food Science in May 2014 in Food Science at Louisiana State University. Dr. Jie published three referred articles and 3 abstracts. Dr. Jie was awarded second place at the 2013 IFT- Refrigerated and Frozen Foods division graduate student paper and won the IFT Gulf Coast Section scholarship for his work in seafood. He received economic development scholarship from Louisiana State University in 2014. He worked in novel fish oil extraction methods and seafood processing improvement. In August 2014, he joined Green Pasture Product as the quality control and product safety manager.