You've heard of ketosis, the benefits associated with it and perhaps, have delved deeper into the science of how and why the ketogenic diet works. Maybe you’ve experimented with ketosis yourself. You might even know a bit about what ketones are and how the body uses them.
While the blanket term “ketones” gets thrown around a lot in the scientific and health press, not all ketones are the same.
One ketone in particular, acetoacetate, also known as acetoacetic acid (AcAc), is particularly important due to its role in the production of the “other” ketones. The body can also use AcAc in pretty unique ways. Understanding acetoacetate will allow you to understand ketosis on a deeper level so you can take advantage of its many benefits.
Before getting into the nitty gritty of specific ketones like acetoacetate, let's first take a step back and look at ketosis from bird’s eye view. What is ketosis? Operationally, it can be defined as the presence of ketones in the blood (usually at a level above 0.5mM).
When the body starts to produce ketones this means that it is in a state of fat oxidation—turning fatty acids into ketones—rather a state of carbohydrate oxidation and fatty acid synthesis. This is known as “endogenous” ketosis; since you’re relying on your body’s own fuel stores. Don’t get ketosis confused with diabetic ketoacidosis—a dangerous condition experienced in people with type 1 diabetes and less often in those with diabetes mellitus (type 2 diabetes). In this condition, a high amount of ketones build up in the blood, leading to blood acidity.
No matter the route taken, ketosis is the presence of elevated blood ketones, whether produced by the body naturally or taken through a supplement. Three different ketones exist: acetoacetate/acetoacetic acid (AcAc), beta-hydroxybutyrate (BHB) and acetone. Of these, AcAc is the only true ketone body.
What do we mean by "true" ketone body? Let’s get technical for a moment. In organic chemistry, ketones are organic compounds with a carbonyl bond—they contain at least one carbon atom double bonded to an oxygen, with the other two bonds belonging to carbon molecules or hydrocarbon radicals. AcAc contains this “keto” group, while BHB does not.
AcAc is actually the precursor to BHB and acetone, both of which are derived from AcAc metabolism. For this reason, AcAc is sometimes called the “parent” ketone body.
So, if you’re in ketosis, you likely have a decent amount of both AcAc and BHB flowing throughout your blood.
Why does ketosis exist?
It seems to be an adaptive, evolutionary feature developed in order to ensure survival during times of low energy availability or when few carbohydrates were available. The fuel our body burns is dictated by availability; prioritizing glucose first, then fats, and finally (and least of all) protein (amino acids). When our “preferential” energy source (glucose) runs low—during extended fasts, exercise, and/or consumption of a low carbohydrate (i.e. ketogenic) diet —the body begins to break down fat stores into free fatty acids (FFAs) and eventually turns these into ketones.
Unlike fatty acids, ketones can cross the blood-brain barrier and provide the necessary food for our brains, ensuring survival and the ability to function.
Glucose can also cross the blood brain barrier to provide fuel for the brain, but on a low-carb or ketogenic diet, there is limited glucose available from the diet, so the body must make its own glucose through gluconeogenesis. Thus, up to 60% of the brain’s fuel can be provided by ketone body metabolism during conditions of low energy availability. If we had no mechanism to produce ketones, our brain would be left starved of energy when carbohydrate stores are low. Ketone bodies also play a role in glycogen sparing and limiting protein breakdown during times of fasting and/or starvation.1
Acetoacetic acid is a 3-oxo carboxylic acid, the simplest organic acid in the beta keto-acid group, and highly unstable.
While AcAc is generated first among ketones, it’s generally found in the blood at much lower levels than BHB; the ratio is between 4:1 to 6:1,1 and this can change in different disease or nutritional states. Why is BHB more prevalent?
AcAc is less stable than BHB, and can be spontaneously converted into acetone. As stated before, acetone is usually excreted in the breath, so this process is essentially a waste of energy. On the other hand, BHB is more stable and can travel through the blood and cell membranes more reliably. It’s the preferred form for ketone delivery from the liver to other tissues.
Even though it’s found in lower levels, without acetoacetate, we couldn’t experience the benefits of ketosis (since we would not be able to produce BHB). But how are AcAc and the other ketones generated in the first place?
The process by which we create ketones from free fatty acids (FFAs) is known as ketogenesis. Sensing low levels of insulin and glucose, the liver is triggered to produce ketones. It does this by first breaking down a fatty acid molecule into two molecules of acetyl-coenzyme A (acetyl-CoA) which are then condensed into acetoacetyl-CoA. A third acetyl-CoA molecule is then added, yielding a molecule called 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which then splits, regenerating acetyl-CoA and forming one molecule of AcAc.
AcAc can then be reduced to BHB through an enzyme known as beta-hydroxybutyrate dehydrogenase, or non-enzymatically decarboxylated to form acetone, which is then excreted in the breath (this is why “keto breath” is a thing).
While BHB is the primary circulating ketone found at the highest amounts in the blood, AcAc is also present, and both can be taken up by cells and metabolized to produce ATP.
AcAc undergoes three metabolic “fates” once it is produced in the liver. Some of it will be released and enter the circulation, where it can be taken up by peripheral tissues and used as an energy source. However, due to its instability, some amount of AcAc in the blood undergoes conversion to acetone. The third fate of AcAc involves its conversion to BHB, which is more stable and readily transported throughout the body. This process occurs in the liver. BHB is then exported from the liver, delivered into peripheral tissues (heart, brain, skeletal muscle) and then broken down and used in the citric acid cycle (a.k.a to produce cellular energy currency (ATP) in a process known as ketolysis.
Ketones must be transported from the liver because this organ can’t use ketones for energy—it lacks a critical enzyme (Succinyl CoA-acetoacetate CoA transferase) needed to turn AcAc into acetyl-CoA, where it can enter the TCA cycle and be used to generate ATP.
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Achieving ketosis means elevating your body’s production of ketone bodies. Unless you’re taking an exogenous ketone supplement like BHB, the organic acid AcAc is the ketone your body will produce first when entering into ketosis.
Intermittent fasting is a hot topic, a popular “biohack”, and everyone from CEOs to athletes are using it to level up performance. Some do it for weight loss, some do it for the autophagy benefits, others do it for mental clarity and clear-headedness.
Fasting is also a great way to elevate ketone production, since both blood glucose and glycogen decline during extended periods without eating.
This means the body will increasingly rely on stored body fat (and ketone production) as an energy source to fuel the brain and other tissues.
How long of a fast is necessary to achieve ketosis? 12 - 16 hours seems like the minimum, but this will depend on certain factors like physical activity. In one study, healthy men achieved an 82% increase in circulating AcAc after an 18 hour fast.2 Men and women who fasted for 48 hours experienced a 20x increase in circulating ketones.3 In general, the longer you fast, the higher your AcAc (and other ketones) will be. Of course, consult a doctor before engaging in extended fasts.
Maybe most obviously, boosting natural production of AcAc can also occur through the adherence to a ketogenic diet. Keto diets contain 70% - 80%+ of total daily calories from fat, about 10% - 15% from protein, and the remaining minimal amount from carbohydrate (typically under 50g/day).
How long does it take for AcAc levels to rise on a ketogenic diet? The time will vary among individuals, however, a high fat-low carbohydrate / ketogenic diet for as short as four days has been shown to increase endogenous ketone body availability, increasing levels to around 1mM - 2mM.4 A recent study in military personnel indicated that it took an average of just three days to achieve nutritional ketosis after following a ketogenic diet (containing about 25g of carbohydrate/day).5
Need help formulating your keto diet? We have all the resources need to construct the perfect keto diet for you.
Achieving ketosis requires substantially reducing blood glucose and stored glycogen levels.
Prolonged exercise can achieve both, and numerous studies have shown that exercise is able to increase ketone levels in the body.6,7,8 While BHB was often measured instead of AcAc, we know from earlier than AcAc is required to generate BHB, so it can be concluded that exercise is able to raise AcAc levels in humans.
There seems to be a wide variation in the post-exercise ketone production seen in studies, and this may be due to the nutritional status of participants. For instance, the highest ketone levels after exercise were observed in two marathon runners who had undergone dietary carbohydrate restriction.6
Adding carbohydrates to the diet can prevent post-exercise ketosis.
This suggests that carbohydrate (energy) status of the body is important in determining whether you’ll truly be in ketosis after exercise. Diet might make all the difference.
For this reason, exercising with low-carbohydrate availability is one strategy to achieve ketosis. Another strategy might be to withhold carbohydrates after exercise; both will result in lower blood glucose and promote ketone production.
Nutritional ketosis as a result of a keto diet is often preferred for those looking to lose weight, improve metabolic health, or enjoy the consistent energy benefits obtained through fat utilization. However, ketones can also serve as fuel source for mental and physical performance. In this way, exogenous ketones can serve as a supplemental source of fuel independent of a ketogenic diet.
Few studies have evaluated the effects of exogenous AcAc consumption in humans; many use the more bioavailable BHB ester to induce ketosis. Ketone monoester BHB can boost blood BHB levels up to 3 - 5mM within 30 minutes post-ingestion.9,10
Exogenous supplementation with a 1,3 butanediol acetoacetate diester ketone ester effectively induces ketosis within 30 minutes (as assessed by blood BHB) and sustains ketosis for up to 8 hours in rats.
Levels of ketones following AcAc ingestion have been reported to exceed those achieved with a ketogenic diet or starvation.11 Consistent supplementation with this ketone ester has been used to achieve ketosis for a period of 4 weeks.
One study has evaluated the effects of AcAc ingestion on cycling performance in athletes. While consuming a ketone diester (Acetoacetic ester) increased serum levels of AcAc, performance was impaired over the 50 minute time trial, indicated by a 3.7% reduction in power output and reduced cadence.12 Gut discomfort and intolerance of the ketone ester were cited as possible reasons for the performance decline.
Studying the effects of exogenous acetoacetate shows that it's a ketone body with wide-ranging benefits throughout the body that might be unique to this organic compound.
Ketones are more than brain food—they might provide protection against certain disease and central nervous system afflictions.
In one study, treating neurons with 5mM of acetoacetate protected against cellular toxicity induced by glutamate—a neurotransmitter that contributes to neuronal degeneration and disease.11
AcAc might even have superior neuroprotective properties to the more well-known BHB. In mice, administration of an acetoacetate diester delayed central nervous system oxygen toxicity and increased time to seizure, a result attributed to increased AcAc levels rather than BHB.11 The same ketone ester was shown to improve motor coordination, learning and memory, and brain synaptic plasticity in mice with a rare neurological disorder.13 Ketogenic diets have been used for decades to treat epilepsy, and these newer studies are providing evidence that exogenous ketones might also have benefits for the brain in other conditions.
Macrophages are inflammatory mediators in the body which coordinate tissue repair, remodeling, and fibrosis after cell injury. Interestingly, macrophages have the capability to utilize many sources of fuel for energy—including ketone bodies. AcAc can be metabolized by liver macrophages, and was shown in one study to protect mice against diet-induced liver fibrosis and injury.14
A novel function of AcAc might be as a signaling molecule in the muscle. Studies have indicated that AcAc promotes the proliferation and regeneration of muscle satellite cells and recovers muscle strength, muscle integrity, and improves exercise performance in mice.15 AcAc also reduces the rate of glucose uptake and glycolysis in skeletal muscle,16 evidence for a possible performance-enhancing effect of ketone body metabolism due to glycogen sparing during exercise.
How do you know if you’re truly in ketosis? While several different methods exist for measuring ketones, certain tests are specific to each.
To measure acetoacetate, urine testing is the preferred method. This is because AcAc “spillover” is present in the urine. For this reason, AcAc levels can be tested using commercially available urine testing strips and completed at home or...anywhere.
Performing the measurement involves placing the end of a test strip through a sample or stream of urine and removing it immediately. After about 15 seconds or so, the test end will begin to change color. The ketone color chart provided on the test strip container will inform you of the amount of ketones detected (if the test is positive). The levels will range from trace, moderate, to large amounts.
Is urine testing accurate? Most studies conclude that urine testing accurately estimated AcAc levels. It is suggested that you test for ketones in the early morning or late evening after dinner in order to obtain the most reliable measurement.17,18
Advantages of urine testing include the fact that it’s relatively inexpensive, commercially available, quick, easy, and convenient.
Want to check your ketone levels on an airplane? No problem.
There are, however, a few issues with urine testing. For one, the test strips give only an approximate estimation of ketones rather than a quantitative number. Certain medications and vitamins (vitamin C) can lead to inaccurate results (false positives and negatives), and even hydration levels can influence the results of the test.
Lastly, as you keto adapt, urine testing may become progressively more inaccurate. After you’ve been on a ketogenic diet for some time, your body becomes better at using ketones for energy, metabolizing more and excreting less.19 You may even begin to conserve more ketones and directly utilize fat as fuel for some tissues. As a result, there will be less AcAc in your urine and the test strips might show a negative result, despite you having a high amount of blood ketones.
Nevertheless, urine testing can be a semi-quantitative and valuable way to test for ketones in the initial phase of a ketogenic diet to determine if the protocol you are following is working. The “gold standard” method used in most research studies involves direct measurement of ketone bodies like AcAc using gas-chromatography mass-spectrometry to separate, identify, and quantify the amount of organic compound present. Have a spare spectrometer at home? Be our guest. Otherwise, stick to the strips.
There are other ways to test for ketosis specific to the metabolite of interest. Breath ketone meters are a product new to the market. These devices measure the amount of acetone in the breath (remember, acetone is the waste product generated from the breakdown of AcAc). Breath ketone meters may be most accurate when measuring low ketone concentrations such as during a ketogenic diet. For exogenous ketone supplementation, when ketone levels spike quickly, breath acetone isn’t as accurate a reflection of blood BHB.
One more way to test for ketosis is through blood testing—usually through the use of a BHB / glucose meter. While requiring a finger stick, this measurement gives the most accurate reading of blood ketone levels. It might be best to experiment with testing methods to see which works best for you and your lifestyle and diet.
Much attention is given to ketones, and BHB seems to get the spotlight. While there are some good reasons for this (it's easier to measure and found in the highest concentrations in the blood), this doesn’t mean AcAc is any less important. In fact, as we’ve seen, all keto-dieters should now know that AcAc is essential for ketosis to occur. Without it, we couldn’t boost blood BHB (endogenously, at least) and get the benefits that come with it. It’s not often used as a supplement, but AcAc has many benefits for the brain, liver, and muscle.
Understanding ketone body metabolism, where AcAc comes into play, and how this ketone body influences overall metabolism is crucial to understanding your body and how it operates while in ketosis.
Knowing the proper ways to measure for acetoacetate, you can begin to formulate your ketogenic diet, supplementation plan, and testing regimen to optimize your time in ketosis—reaping all of the benefits ketones have to offer.
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