Part 3 – Omega-3 Index Series: What Factors Influence the Omega-3 Index?
In theory, we’d love to be able to predict your Omega-3 index with 100% certainty if we know, in addition to how much fish and supplements you consume, your age, your sex, your race, your ethnicity, your body weight, whether you smoke or not, and some other genetic factors. But it’s a little more complicated than that.
The Omega-3 Index as a Biomarker
First, let’s go back to the Omega-3 Index’s role as a biomarker. In a 2007 study from Metcalf and colleagues in Australia, they took pieces of heart tissue at surgery from 28 patients who had been treated with about 6 grams of omega-3 fatty acids a day for between seven and 70 days before they went into surgery. The study started at the beginning of supplementation and then by the time of their surgery they had been taking it for between seven and 70 days.
The researchers were able to look at the rate of incorporation of EPA and DHA in the red cells and into the heart. What they found was that the increase in EPA and DHA in the heart correlated nicely with the increase in EPA and DHA in the red blood cell.
BLOG: Discovering the Omega-3 Index
But Dr. Harris and his colleagues were curious about other tissues, so he collaborated with Dr. Fenton at Michigan State University, who did several studies in mice, feeding them different levels of omega-3 and then comparing the red blood cell to other internal organs and their omega-3 content.
She found that there was always a nice relationship between the red cell and tissues, regardless of the tissue type looked at. So that gave Dr. Harris further confidence that the Omega-3 Index is a good marker of the internal omega-3 status of the organs.
Genetics & Lifestyle Factors Influence the Omega-3 Index
Dr. Harris and colleagues did a study in the context of what’s called the Framingham Offspring Study. This is a large population of people in Framingham, Massachusetts, who have been studied very carefully for a long time. In this case, they measured omega-3 levels of roughly 3000 people at the beginning of the study.
BLOG: 3 Reasons to Measure Omega-3 Levels in Red Blood Cells
Of course, there was a quite a bit of variability in the omega-3 levels among those studied, so the question was, “Why does one person have a high level and another a low level? What are the factors that control it?”
So they looked at what’s called covariates, which are other factors that might affect the Omega-3 Index. In the graph below, they are trying to explain 100% of the variability, which would be 1.0 on the vertical axis. Reaching 1.0 would mean that with what we’re able to understand and explain we could account for the full range of variation between people.
They don’t reach it, unfortunately. They accounted for non-genetic factors, or lifestyle factors, such as smoking, omega-3 intake, fish oil supplementation, etc. They also include age and sex, and although sex is genetic in a sense these are demographics that are not specifically related to a given gene. These factors only explained half of the variation between people.
This is called the “offspring” study because it involves the descendants from the original Framingham Study, which started in the 1940s. The offspring study started in the 1970s and consists of many people who are related to each other — brothers and sisters, cousins, and so on. So, researchers were able to take advantage of the fact that there was a heritability or a relatedness variable in this study that helped Dr. Harris and colleagues tease apart how much genetic or non-genetic factors explained.
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There was roughly 25% variability associated with genetic factors. After that they considered the non-genetic factors, which accounted for a further 45% of variability and left about 30% unexplained. Dr. Harris says he doesn’t know what specific genes are at work here, he just knows there’s some genetic connection because the more closely you are related to someone, the more similarly you will respond. This remains early research, but the point of this example is that there’s a lot of factors influencing your Omega-3 index and we don’t know what all of them are, which is why you can’t make a complete prediction. The only way to know your omega-3 level is to measure it.
Validity of the Omega-3 Index Dried Blood Spot Test
We’ve talked a lot about how blood omega-3 levels correlate with tissues, as well as the variability and utility of the Omega-3 Index test. Now let’s talk about how OmegaQuant’s dried blood spot Omega-3 Index test correlates with the red blood cell tests, its stability, its reproducibility or repeatability in the laboratory, how much blood you need to take, and whether you need to be fasting or fed. All of these factors are important to laboratories like OmegaQuant when measuring Omega-3 status.
The graph below shows a comparison between the EPA and DHA level measured from a dried blood spot and the level given by the red blood cell Omega-3 index. This data is from about 100 people in fasting or fed states.
We’ve defined the Omega-3 Index as a measurement derived from red blood cells alone. However, our dried blood spot is a dried sample of whole blood, which contains a mixture of both cells and plasma. Since these portions have different omega-3 levels, you’re not going to get the same number from a spot of whole blood vs. just the red blood cells. Still, there is a very strong correlation. In other words, if we know what the dried blood spot EPA and DHA level is, we can very accurately predict what the red blood cell Omega-3 Index is. And that’s what the study above was able to show Dr. Harris and his team, that they correlate very well.
BLOG: What Do Your Omega-3 Index Results Mean?
Now let’s talk about just how much blood you need to get an accurate Omega-3 Index score. The image below shows that we can measure the Omega-3 Index accurately in as little as two microliters of blood, which is a very tiny amount. Note here that the optimal amount for a dried blood spot is a full drop of blood, which is around 25 microliters. If you just let blood drop off your finger and don’t touch it to the paper, that will give you roughly 25 microliters. Sometimes we do not get that, and so we like to be sure that even if we have very little sample to work with, we can still get an accurate answer. The figure below shows increasing amounts of blood dried on a piece of paper, ranging from two microliters to 50 microliters of blood.
Dr. Harris captured different amount of blood with two different blood samples. One set of blood samples was relatively high in omega-3 and one set of blood samples was relatively low. And they measured the Omega-3 Index in two microliters, eight, 10, 12 and up to 50 microliters. As a result, they got the same Omega-3 Index regardless of how much blood was in the drop. And it is not dependent on having a relatively low or high level of omega-3s. They were able to measure the Omega-3 Index accurately in both settings. This gave them confidence that they could use a dried blood spot almost regardless of how much blood people put on their sample collection card. But keep in mind, a 25 microliter drop is ideal.
One factor that can affect a dried blood spot omega-3 level that doesn’t affect red blood cells is how much fat or how much fatty acids are actually in the plasma, and that varies with how much fat you’ve just eaten. It’s possible that a blood sample taken in the non-fasting state right after a big meal might give you a different Omega-3 Index than a blood sample taken after an overnight fast.
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So Dr. Harris and colleagues performed an experiment with 16 healthy volunteers. First they collected dried blood spot samples, a blood drop taken with a little fingerstick before a meal. Then they were given a large breakfast that contained on average about 800 calories and 43 grams of fat. That’s roughly half the amount of fat that a person typically eats in a day. So it’s quite a high-fat meal. It’s not really a typical breakfast that people eat in America but they wanted to press it.
Then they took a second dried blood spot collection about three and a half hours after the meal, which is typically when the fat content reaches its peak in the bloodstream. This sample would reflect the period of greatest effect on the plasma fatty acids as a result of this meal.
When they measured the Omega-3 Index before and after the meal what they found was that there was a little reduction in the reported Omega-3 Index. That’s because, again, the Omega-3 Index is a percentage, namely EPA and DHA, as a percent of all the fatty acids. So, if you add other fatty acids to the blood sample the percent that’s EPA and DHA will go down a little bit. But it didn’t go down much — less than 4%. On average the Omega-3 Index before the meal was 6% versus 5.8% after the meal, which is a very tiny difference. So what this shows is that even a dried blood spot collected in a relatively extreme fed state still gives you a very good representation of the true red blood cell EPA and DHA level.
The Reproducibility of the Omega-3 Index
One of the important metrics we always look at in the laboratory is reproducibility of measure. In other words, if you measure the same blood sample repeatedly do you get the same answer? If your assay doesn’t give you the same answer on the same sample, you’re in trouble. If it does, that is good and what you expect. And so, Dr. Harris tested the Omega-3 Index measurement from dried blood spots in one of the most challenging ways they could imagine — a field study.
In this study, dried blood spots were collected from women in communities in two or three states in Mexico and sent to OmegaQuant. The variables in this experiment included not only the variability that is always present within a given analytical method, but that blood samples were collected on filter paper with a various amount of time between getting the blood spot to the researchers in Mexico and then those researchers shipping them to Sioux Falls, SD, for analysis.
VIDEO: Why You Can Trust Your Omega-3 Index Score from OmegaQuant
They also tested what’s called blind duplicates. Basically, these are blood samples taken twice from the same person but labeled differently. So, in the laboratory they didn’t know which sample was the real one, and which was the duplicate. After they analyzed all of the samples, they went back and looked at which were the duplicates to see how closely they matched each other. If they’re very close that’s good.
Dr. Harris and his team were also able calculate what’s called the coefficient of variability for the Omega-3 Index. A coefficient of 15%, meaning a 15% variation of reproducibility, is considered acceptable. If the coefficient is under 5%, that’s very good. You don’t want to use tests that have really high variability. The Omega-3 Index comes in at just under 5%, which is excellent.
Another important feature of the way blood samples are collected at OmegaQuant is the antioxidant cocktail added to the sample collection card called F-A-P-S, or fatty acid protection system. This is a solution containing several antioxidant compounds that protect the polyunsaturated fatty acids, such as the omega-3s and omega-6s in the dried blood spot, from becoming oxidized while this sample is in transit to the laboratory.
If you don’t treat the paper that you put the blood on with an antioxidant, the blood will oxidize and degrade. The experiment below looked at dried blood spot samples using treated and non-treated filter paper. Dr. Harris and his team analyzed the same blood sample put on two different cards, over a period of days, taking the average of three tests in each case.
The cards on the blue line above were pretreated with OmegaQuant’s antioxidant solution. This same sample was placed on cards without any pretreatment, allowing it to be subject to oxidation from the air. What they found was that the Omega-3 Index was quite stable, certainly over the first four or five days, which is typically how much time it takes to mail in a sample.
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When the fatty acid protection system was not on the sample collection card, then the fatty acids started to degrade. By day seven the untreated card hit the unacceptable limit of 15% loss. The treated card stayed well within the acceptable range, even up to 44 days, and it never takes that long to mail a card in. Typically, it is four to six days until we get the cards. But the antioxidant treatment did protect it from reaching this limit of 15% loss, while that lower level was hit in a week with the untreated card. So it’s very important to use properly treated dried blood spot collection cards to ensure accurate results.
Omega-3 Index Regulatory Status & Research Collaboration
Regulatory issues are very important for this test, and for any blood test that physicians order on patients and which patients or consumers can order themselves. Our laboratory is what is called CLIA certified. CLIA stands for Clinical Laboratory Improvement Act. Its legislation was established by the US Congress to ensure that medical laboratories that are reporting results to patients have certain quality features about them, and you have to meet certain performance metrics in order to maintain certification. OmegaQuant been certified for the last 11 years.
We have also approached the US Food and Drug Administration (FDA) about getting our tests approved. After their evaluation they said we really don’t need to have the test approved by them and that it’s clearly not invasive enough to be worrisome. They told us we are not going to hurt anyone doing this test, and that we could offer the test directly to consumers, without pre-market review or without post-market regulatory follow up requirements.
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So, we feel comfortable that we have, if not the blessing of the FDA, the opinion of the FDA that they don’t need to regulate this test. We also have approval in the European Union, as well as Australia, for the dry blood spot collection kits, which are registered as a medical devices so they can be sold in those countries. And we’re working with other territories like Asia to get regulatory approval as well.
Another highlight that really sets OmegaQuant apart is its research collaboration. Dr. Harris and his team have collaborated with more than 100 research institutions over the last 10 years, where they have been put in the position of analyzing fatty acid blood samples. These organizations have trusted OmegaQuant with this analysis, because they have been able to do high quality work and publish alongside them. OmegaQuant’s research partners are come in all different shapes and sizes and include pharmaceutical companies, universities, and institutions like the National Institutes of Health. OmegaQuant has also worked with the Framingham Study, The Women’s Health Initiative Memory Study, the US Army and many more.
One reason these research organizations look to OmegaQuant is because they know they can get an omega-3 number from a variety of laboratories, but what they cannot get is the Omega-3 Index. Our test is unique to our laboratory and our method, and it’s our method that’s been used in several hundred studies.
But we don’t just provide a number like some labs would do. We provide expertise and depth in the understanding of omega-3 and omega-6 fatty acid biology. And many investigators that we work with don’t really have much expertise in that and they rely on us to help them understand what the numbers mean. This is why Dr. Harris and his daughter, Dr. Kristina Harris Jackson, have been authors and co-authors on hundreds of papers resulting from this work. This work keeps Dr. Harris and his team excited about meeting its mission, which is to advance the science of omega-3 fatty acids for human health and reduce the risk and burden of disease. The long-term goal for OmegaQuant is to get the Omega-3 Index recognized as a risk factor for heart disease in the same way cholesterol is. Until then, the Omega-3 Index is a minimally invasive way for doctors, patients and the public to get more information about their fatty acid status.
INFOGRAPHIC: A Brief History of Omega-3 Research