There is a little miracle of science happening in your body right now. As you read this, a minuscule 5 grams of a high-energy molecule called adenosine triphosphate - ATP - is causing all kinds of reactions in order to give you the energy to sit at your computer. In total, 8 ounces of ATP is being recycled hundreds of times each day, so many times that a human can use their body weight - 200 pounds of ATP in my case – every 24 hours.
ATP is not stored and summoned as needed, it is created on the go. This science miracle happens in tiny energy factories called mitochondria. Mitochondria are rightly called the energy factories of cells because they take energy contained in food and transform it into something our cells can use.
It’s easy to imagine that in a system so vital, when things go wrong they go really wrong. Figuring that out how to make the wrong things right again has been the goal of science since the discovery of ATP by researchers in 1929, especially once it was realized that mitochondrial pathologies are at the root of some diseases even though the damage is not mitochondrial.
Confusing, right? Imagine being a scientist and having to try and figure all that out. For decades, there was so much information available about mitochondria and its impact on disease and aging ... except what is inside a ‘black box’ in the mitochondrial matrix, where everyone knew what must be happening but couldn’t find a way to make a positive difference. ‘Positive’ will turn out to be a pun, as you will see by the end of the article.
To solve the mystery, I interviewed two scientists on the leading edges of the field at various times: Dr. Michael Murphy of Cambridge, recent co-inventor of a molecule called MitoQ, which made external antioxidants truly bioavailable for mitochondria; and Professor Frederick Crane, who discovered the body's natural antioxidant, Coenzyme Q, in 1957.
Practically everyone has heard of antioxidants by now. They are the broad category of foods or supplements that prevent cell damage, and include things like Vitamin C and Vitamin E. Antioxidants prevent oxidative stress from another reaction everyone has heard about - free radicals, molecules formed when we convert food into energy or when we exercise. The same process of reduction and oxidation that gives us energy can damage cells, and that damage has been linked to everything from cancer to Parkinson’s disease.
Preventing damage is key to preventing disease. But it isn’t as simple as taking an antioxidant or eating more meat.
Mitochondria have two obstacles designed to help insure that the only thing happening in its factory is what nature decided is supposed to be happening – an inner and outer membrane. The outer membrane is porous enough that molecules can get through, and so antioxidants have had little trouble there. The inner membrane is much more restrictive because it protects the mitochondrial matrix, the proteins that will give us energy. Inside that matrix is the machinery that helps run an electron transport chain in the mitochondrial inner membrane, which is just what it sounds like – electrons traveling from one protein to the next. That electron transport chain creates what is called the "molecular unit of currency" in cellular energy - ATP.
When the problem is inside mitochondria, such as in mitochondrial diseases, nature needs a little help.
There are helper molecules that give biochemical assistance throughout this mitochondrial process, called cofactors in biochemistry but more commonly known as coenzymes. And one coenzyme in particular became famous because it was found to exist in a completely oxidized form or a completely reduced form in humans, which means it can function as part of the electron transport chain and also act as an antioxidant. It’s called Coenzyme Q10 and it paved the way for understanding how ATP was made, which got Dr. Peter Mitchell a Nobel prize in chemistry in 1978 . It also led, over four decades later, to a molecule named MitoQ that figured out how to get mitochondria functioning in older people as it does in younger ones. Like the electron transport chain itself, the link between the work of Crane and Murphy, 40 years apart, can be shown rather clearly once you know where to look.
To learn about CoQ10, we have to go back to the University of Wisconsin–Madison Enzyme Institute in the 1950s, where Dr. Frederick Crane was a young post-doctoral researcher working on one of the most difficult problems in science; the biochemical pathways involved in how mitochondria in cells produce energy. Crane is now Professor Emeritus at Purdue and semi-retired at his home in Illinois. He still writes papers, he still keeps up with the latest research, and he remembers his time at the Enzyme Institute like it was yesterday.
Born in 1925, Crane took an unconventional path to unlocking the secrets of antioxidant defenses in humans – he was a plant physiologist studying niacin synthesis who started out as a chemist but got interrupted by World War II. That’s a road less traveled for most scientists.
He got his doctorate from the University of Michigan and, like many researchers at the time, wondered what he could work on next. We communicated via phone interviews and emails and he explained that early history and how he ended up in Wisconsin.
“My advisor at the University of Michigan, Professor Gustavson, said he liked what D.E. Green was doing in metabolism at the Enzyme Institute so I wrote to him and said ‘I would love to study metabolism’. David replied that he was studying energy coupling, not metabolism, but if I might love that too he would send an application,” Crane said.
Apply he did. Crane is a modest sort and says there were probably few people interested in that kind of job, which made it easier for him to get. He also recounted his concern about his abilities because he had limited success at first, to a point where he became worried about his future.
Prof. David Green’s group at the University of Wisconsin-Madison Enzyme Institute in 1956. Back row (left to right) Dave Gibson, Joe Hatefi, Tony Linnane, Dexter Goldman, Nat Penn, Bruce Mackler, Howard Tisdale, Al Heindel, and Dan Zieglar. Second row (left to right) Seishi Kuwahara, Salih Wakil, Helmut Beinert, Bob Lester, Alton Frost, Johan Jarnefelt, David Green, John Porter, Elizabeth Welch, unidentified, Wanda Fechner, Bob Basford, unidentified, Fred Crane, Sedate Holland, Carl Widmer, Robert Labbe, and Edward Titchne. Front row Ruth Reitan, Amine Kalhagen, Cleo Whitcher, Elizabeth Steyn-Parve, Jean Karr, Joanne Gilbert, Mildred Van der Bogart, Mary Benowitz, and Irene Wiersma. Credit: Fred Crane
“My lone accomplishment at the Enzyme Institute had been one purification of a batch of aldehyde oxidase, a well known enzyme , easy to purify. I did extract an NADH dehydrogenase from mitochondria and purify it, but credit for that was assigned to another post-doc. Meanwhile, other researchers had done some good work.”
And then an intellectual guillotine appeared over his desk. Green stopped by and asked Crane if he had thought about where he was going to work next year. As any post-doctoral researcher, or any employee anywhere, can tell you, that question is a ‘you should find a different career’ hint, and so he began writing letters looking for a new job. And though the climate for unfunded academic plant physiologists was difficult, he did get an offer, from a researcher at Caltech.
But in the month between Green’s thunderclap of a career-defining question and the job offer, a thunderclap in biochemistry happened: Crane found a new enzyme, the electron transport flavoprotein (ETF), an entirely new function for a flavoprotein and a crucial waypoint on the path to discovering the secret of how mitochondria produces energy.
Suddenly, they knew they were onto something important. Green, in a bit of a mea culpa, told Crane that a post-doc should stick around for at least 10 years, and said he hoped Crane planned to stay at the Enzyme Institute and not take the Caltech job he had written him a reference letter for.
How he went from soon-to-be-unemployed post-doc to the toast of the field is a fascinating story. In the cases of both Crane and Murphy, their breakthroughs were what they called a little bit of luck but that were really brought about by some non-traditional thinking.
“That was an example of showing persistence in the face of lost hope and making a major discovery in the face of catastrophe,” Crane deadpanned.
As it turned out, his perceived weakness had been an advantage – he only knew about plant physiology. At Michigan he had studied hydroquinones as terminal oxidases in plants but animals didn’t have quinones, experts said. Since he wasn’t an expert he decided to learn about animals and quinones the hard way. Everyone was trying to figure out mitochondria and Crane now had access to dual beam spectrometers and ultra centrifuges. And cauliflower.
“Every Saturday I brought my lunch and a couple of cauliflower heads and spent the afternoon and often part of the evening to try and look for something like Vitamin A, because our analysis on hearts we had done showed that mitochondria has almost all of the vitamins you could think of except Vitamin A. I didn’t find any but quickly noticed that cauliflower mitochondria were yellow compared to beef heart mitochondria which were brown.”
So he decided to look for carotenoids in the extract. By looking for carotene in cauliflower on Saturdays when no one was around he came up with the idea to look for carotenes in beef hearts. And he found three of them, but he found something else also, what would come to be known as Coenzyme Q.
Animals didn’t have any quinones, it was said, and common knowledge of organic spectra told him what he was seeing couldn’t be what he was seeing in the unknown yellow compound. Since he was not an expert, he had borrowed a book from the library on organic spectra by R.A.MORTON. It said quinones had an absorption peak of 265. “Since CoQ has a peak at 275 it didn’t fit very well but as a plant physiologist, I kept thinking this ‘must be a quinone’.”
But it didn’t look good at the time. All he had was an inactive flavoprotein to show for his work, even if he knew he was close to something. He took a shot at purifying the inert yellow stuff because they had already reserved some time on an electrophoresis machine at the medical school. Electrophoresis provided three fractions, one with a big yellow peak and two small light yellow peaks. Crane assayed the three and none showed any activity over the indophenols (blue dye) control. “I have never liked the indophenols assay and one of its problems is variable control,” said Crane. “But the lack of activity in the fractions was clear. With nothing better to do, I pippeted a small amount of fraction 2 into the cuvette with fraction one, which was still blue since no activity was there. Immediately the blue color started to disappear and I got the impression that something in tube 2 turned on the enzyme.
“I washed it to make sure it was not a metal, like iron, I substituted different electron acceptors and made sure it did not react with oxygen, I boiled it to make sure it was an enzyme. We had found ETF, and obviously that was just the beginning of the story, we worked on mitochondrial lipids next. Two years later I found Coenzyme Q and I ended up staying at the Institute for seven years.”
What is intriguing is that beef hearts,which are highly regarded now for CoQ content, weren’t considered valuable at the time. Livers were the default for mitochondria study.
“We used beef hearts because Green believed if you want to find something, start with lots of it.Beef hearts were also free from Oscar Meyer whereas rats cost money. Other labs were killing a few rats every day and getting a tiny bit of mitochondria, so they couldn’t do much fractionation.”
The idea they pursued to figure this all out had been to break up the mitochondrial membrane using detergent and then put the pieces back together. It was truly basic research and likely a dead end so it couldn’t be an expensive endeavor, thus the benefit of free beef hearts. Other biochemists had said beef hearts were a dead end because the Phosphate/Oxygen Ratio was less than 1.
“When we reached a ratio of 2 David brought in a gallon of Mogen David wine and we had a party in the library –even though alcohol was prohibited.”
Celebrating a science breakthrough with Mad Dog 20/20, the official wine of hobos everywhere? Much like today, the post-docs doing the real work in academic labs of the 1950s were clearly not getting rich.
A year after his discovery in 1957, Merck became first to test humans for CoQ and his finding led to new thinking about oxidative phosphorylation, which rapidly accelerated research. By 1961, Peter Mitchell had come up with the Nobel-winning chemiosmotic hypothesis that figured out how ATP was made. Due to all that his breakthrough resulted in, Crane is rightly regarded as a true pioneer in the field of bioenergetics.
There was also a personal bonus in the circuitous path he took to discovering CoQ10. Crane’s freshman year at the University of Michigan was interrupted by being drafted and sent off to fight in World War II. He was discharged in 1946 and returned to Michigan by train to resume his college career – he met his wife on that ride.
Cut to the 1980s, when pure CoQ10 was commonly available in large quantities, its reduced and oxidized cycle was well understood and there were methods to easily measure blood concentrations. CoQ10 was heralded as the bane of free radicals,supplements were everywhere, and clinical trials sprang up to investigate how CoQ10 might benefit people with cardiovascular diseases and even cancer.
And then . . . nothing.
It didn’t work, at least not the way it needs to work if venture capitalists are going to throw big money at it. CoQ10 instead became solely a dietary supplement.
There was always a belief that bioavailability was the missing piece of the puzzle.Various techniques had been tried to increase the bioavailability of CoQ10. Without uptake, perhaps taking a CoQ10 supplement was no better than eating meat or fish to get antioxidants.
Earlier in this piece is mention of a tough inner membrane. Lacking some sort of nanotechnology, getting enough antioxidants inside the mitochondrial matrix was difficult. That breakthrough happened in 2001, when Dr. Mike Murphy and Professor Rob Smith cracked the code.
Since mitochondria, our cellular energy factories, use the oxygen we breathe to turn fat and carbohydrates in food into ATP, mitochondrial diseases are usually due to oxidative damage, disrupted energy production, or out-of-control calcium homeostasis, notes Dr Murphy. But that’s not why he and Smith set out to create the mitochondria-targeted antioxidants which resulted in MitoQ, the first such compound to undergo clinical trials in human patients.
Like with Crane and the original discovery of Coenzyme Q10, though everyone knew what had to happen, it wasn’t trivial getting there and, at first, it wasn’t even what they were trying to do.
The belief about the lack of effectiveness of CoQ10 for therapy was that not enough antioxidant became bioavailable. Murphy and Smith jumped that hurdle by using the same ubiquinone as found in Coenzyme Q10 but leveraging the voltage across that difficult mitochondrial inner membrane to get more of it inside mitochondria. They did it by using “lipophilic triphenylphosphonium cation”, a positively charged salt connected by an alkyl chain to the ubiquinol, to bypass the biological wall. It resulted in an antioxidant that could quickly be reduced to active quinol form by cells,but at levels up to 1,200 times greater than CoQ10.
It sounds simple. And yet it wasn’t. People had been trying to leverage physics to get better uptake into mitochondria for decades. The body is breaking down chemicals in food into energy and much simpler elements, protons and electrons, just as would happen if we set spaghetti on fire but in a slow, measured way. The process is even similar when it comes to releasing oxidation products like carbon dioxide and water. Antioxidants weren’t working because they weren’t impacting mitochondria in a meaningful way. Getting enough might mean so many vitamins the effects could be toxic.
MitoQ solved that by leveraging the charge differentiation in mitochondria, the machinery that turns food into electrons and protons. The evolution of it is intriguing because they weren’t trying to solve that problem, they were just trying to see what it was.
“The charge differentiation was originally a delivery mechanism because we wanted to know what was happening inside the mitochondria. We wanted a probe that was going to report on what was happening,” Murphy said. “Then when it worked the next idea was, ‘why don't we make a therapeutic molecule like an antioxidant?’ So we created a targeted version of Vitamin E and published that in 1999. And then we came up with MitoQ,which worked better for various reasons.”
Accumulation of MitoQ10 into cells and mitochondria. MitoQ10 will first pass through the plasma membrane and accumulate in the cytosol driven by the plasma membrane potential (Dyp). From there it will be further accumulated several-hundred fold into the mitochondria, driven by the mitochondrial membrane potential (Dym). There it will be reduced to the active antioxidant ubiquinol. In preventing oxidative damage it will be oxidised to the ubiquinone which will then be re-reduced. ( Murphy, MP, Smith, RAJ, 'Targeting antioxidants to mitochondria by conjugation to lipophilic cations', ANNU REV PHARMACOL TOXICOL Volume: 47 Pages 629-656 2007)
Prominent among those reasons was the length of the carbon chain that connected the ubiquinol and the phosphonium. Why try a 10-carbon tail, I asked, because that can’t have been the ideal solution for either a biochemist or an organic chemist?
“It's a funny story,” Murphy said. “The Vitamin E compound we made had a 2 carbon tail and that was reasonably effective. MitoQ, as you know, has a 10-carbon tail and chemically that makes it a bit unpleasant to work with. It's hydrophobic – oily- which is not so great. We started with a 10 carbon chain for chemical reasons, because the precursor of a quinone with a 10 carbon chain was something we could use chemically on the other end was commercially available so we just bought that.”
Like with Crane and Green and the fact that Oscar Meyer hot dogs did not use beef hearts for some reason, sometimes the best science gets done because you have to be creative with what you can get your hands on.
“So we really started with a 10 carbon chain because we had a precursor we could buy, idebenone. We made the 10 carbon one and it worked beautifully but Rob and I felt like we had just started with that for arbitrary reasons and the 10 carbon chain is a bit of a pain to use, it's difficult to crystallize, and so after we were successful we still decided to go back and try different chain lengths and find the optimal one. We made various chain lengths - 7, 5, 3, 2 and a 15 as well –and after we did all that, the 10 carbon chain turned out to be far better.
“We got lucky the first time, which is rare.”
So why did the 10 carbon chain turn out better than 7 or 15?
“You have a balance between the charge in the 10 carbon chain which gives it a fair amount of hydrophobicity, so it's quite oil soluble and that means that it goes across membranes very rapidly.”
But that’s a bad thing, ordinarily, when you are trying to control mitochondria, right? Those membranes exist for a reason.
“It's more tricky chemically,” Murphy said, “it's oilier, but it's soluble enough in water that we could get it in. The critical thing was you could get a 500 micromolar solution, which is very soluble - because it's a salt it dissolves quite readily in water. The long carbon chain goes straight through membranes extremely quickly, far quicker than the simple molecule, so that means it gets into cells, into mitochondria vivo, within a few minutes of administration.
“The second thing was that the hydrophobicity meant that once it was taken up by mitochondria it adsorbed, it stuck to the matrix facing surface of the inner membrane so it was where we needed it, which was on the surface of the membrane, to block oxidative damage.
“And then finally it turned out that the 10 carbon chain meant that the quinol and quinone could be rapidly recycled, and if you made a shorter chain it just didn't get to the active site, it wasn't recycled as quickly.
“So we just got lucky but we showed later there was a rational reason and that meant when we made subsequent molecules we could use the same design strategy, and other people have followed us.”
This was a real breakthrough and it led to getting MitoQ into human clinical trials when so many others had failed at the animal stage. Murphy said even he and Smith were surprised at how well it worked. “We weren't sure it would work, we thought in vivo not enough might get in. So showing it got in and that it was protective, those were quite important breakthroughs.”
Venture capitalists were intrigued because it worked well in animal models, so they funded human trials for Parkinson’s disease. There, like so many new compounds, it didn’t pan out. Why? Because animal models are truly a different beast.
“It worked in animal models but lots of things do and that's because you can get it in early,” Murphy said. “The problem is we can't diagnose that way. If I am going to get Parkinson's in 20 years, I should start taking the compound right now, but we have no diagnosis that can do that and even if you could, you can't really do a clinical trial like that.”
It was proved safe and it did show success, though those data came later, after VCs had lost interest. “It was working in the liver and in animals studies and in transplantation, kidney damage…I think MitoQ is a very good molecule and applicable to a whole bunch of diseases but maybe it has to wait until it becomes a generic.”
That was discussed in The Most Promising Antioxidant You Never Heard Of. Though new VCs aren’t going to embrace a molecule for which they will have to pay tens of millions of dollars to existing VCs just to have the rights to spend millions on new trials, once it is no longer under patent, it may take off again. Just in the last year, half a dozen studies in animals used MitoQ and found positive effects.
Yet, trials or not, people read those studies and they are going to experiment on their own, since the product has been shown safe and can be purchased. Murphy recognizes those efforts but is cautious –“Yes, it’s interesting but it's anecdotal, they aren't properly controlled studies.”
But they are happening, and that has to be part of the story of antioxidants, yet that won’t be the next article. The next article will be getting deep into organic chemistry with Murphy’s partner in the discovery of MitoQ, Professor Rob Smith.