More than a decade ago I spent a lot of time in the lab, trying to preserve kidneys for transplantation. The challenge was not related to rejection, the sexier end of research, but to a more pressing problem. As soon as a kidney, or any other organ, is removed from the body, the clock starts ticking – frantically. In the case of kidneys, decay renders the organ unusable within a couple of days. In the case of hearts, lungs, livers, and so on, time is even more pressing: they can’t be stored for much more than a day before they are wasted. The terrible prospect of rejection sharpens the problem. It is vital, literally, to match the immune profile of the donor organ to that of the recipient, to prevent acute rejection taking place on the operating table before your eyes. That often means transporting organs over a few hundred miles to a suitable recipient. Given the constant shortage of organs, any wastage is a crime. A stride forward in preservation, giving longer to locate the most suitable recipient, to arrange transport, and to mobilise the local transplant team, would waste fewer organs. Conversely, if we could work out exactly when an organ became unusable then we could salvage organs otherwise condemned as irretrievably damaged, for example, those taken from non-heart-beating donors.
It is practically impossible to tell, just by looking at a stored organ, whether or not it will function after transplantation, even if we take a biopsy and scrutinise it down the microscope. When an organ is removed from the body, the blood is flushed out using a carefully formulated solution, and the organ stored on ice. All looks well, but appearances can be deceptive. An apparently normal organ may become irreversibly damaged after transplantation. Paradoxically, this injury is thought to be caused by the return of oxygen. The storage period primes the organ for a disastrous loss of function upon transplantation, caused by oxygen free radicals escaping the mitochondrial respiratory chains.
One day I was in an operating theatre, fixing probes to a kidney during a transplant operation, in the hope of working out what was going on inside without physically taking a sample. The machine we were using was ingenious – a near infra-red spectrometer. It shines a beam of infra-red rays, which can penetrate several centimetres across biological tissues, and measures how much comes out the other side. From this, a complicated algorithm calculates how much radiation is absorbed or reflected on route, and how much passes through. The precise wavelength of infra-red radiation chosen is critical, as different molecules absorb different wavelengths. Choose your wavelength with care, and you can focus on haem compounds – those proteins that incorporate a chemical entity known as a haem group, such as haemoglobin or cytochrome oxidase, the terminal enzyme of the respiratory chains, deep in mitochondria. Not only is it possible to work out the concentration of haemoglobin – both oxygenated and de-oxygenated forms – but you can calculate the redox state of cytochrome oxidase, which is to say you can work out what proportion of cytochrome molecules are in the oxidised versus the reduced state: what proportion is at that moment in possession of respiratory electrons. We paired this technique with a related form of spectroscopy that enabled us to work out the redox state of NADH, the compound that supplies the electrons entering into the respiratory chain. By combining the two techniques, we hoped to gain a dynamic idea of respiratory chain function in real time, without actually cutting into the kidney – obviously an immeasurable advantage during a major operation.
All this probably sounds very sophisticated, but in fact it’s a nightmare of interpretation. Haemoglobin is present in massive amounts, whereas cytochrome oxidase is barely detectable. Worse still, the wavelengths of infra-red rays that the different haem compounds absorb overlap and merge with each other. It can be very hard to tell which is which. Even the machine gets confused. It measures a change in the redox state of cytochrome oxidase when what actually seems to be happening is a change in haemoglobin levels. We began to despair of ever gleaning any useful information from our contraption. Nor did the NADH levels help much. Most of the time there was a fine peak – a high concentration detected by the machine – before transplant, which vanished without trace after the organ had been transplanted, and that was that. It all sounded good on paper but the reality, as so often in research, was uninterpretable.
And then I had my own personal eureka moment, the moment I had my first inkling that mitochondria rule the world. It came about by chance, for one of the anaesthetics being used was sodium pentobarbitone. The concentration of this anaesthetic in the blood fluctuated, and on a few occasions when it did, we found we were picking it up on our machines. The levels of both oxy-haemoglobin and deoxy-haemoglobin remained unchanged, but we recorded a shift in the dynamics of the respiratory chain. Part of the NADH peak returned (it became more reduced) while the cytochrome oxidase became more oxidised. We seemed to be measuring a ‘real’ phenomenon, rather than the usual frustrating noise, because the levels of haemoglobin weren’t changing. What was going on?
It turned out that sodium pentobarbitone is an inhibitor of complex I of the respiratory chain. When its blood levels rose, it partially blocked the passage of electrons down the respiratory chains, and this led to a back-up of electrons in the chains. The early parts, including NADH, became more reduced, while the later parts, including cytochrome oxidase, passed on their electrons to oxygen and became more oxidised. But why did this beautiful response not occur every time? This, we soon realised, depended on the quality of the organ. If the organ was fresh and functioning well, we picked up the fluctuations easily; but if it was seriously damaged it was virtually impossible to take a measurement. We saw the usual disappearance of all the peaks, never to return again. The explanation could only be that these mitochondria were as leaky as a colander – of the few electrons that entered the chain, barely any left it again at the end. Virtually all must have been dissipated as free radicals.
Without slicing out samples and subjecting them to detailed biochemical tests, we couldn’t be absolutely sure about what was really happening in these mitochondria, but we could say one thing for certain – the damaged organs were losing control of their mitochondria within minutes of transplantation, and there was absolutely nothing we could do about it. We tried all kinds of antioxidants, in an attempt to improve mitochondrial function, but to no avail. Mitochondrial function in those first few minutes foretold the outcome, perhaps weeks later – if the mitochondria failed in the first few minutes, the kidney inexorably failed; if they still had some life in them, the kidney had a good chance of surviving and functioning well. The mitochondria, I realised, were masters of life and death in kidneys, and extremely resistant to being tampered with.
Since then, in considering diverse fields of research, I’ve come to realise that the dynamics of the respiratory chain, which I struggled to measure all those years ago, is a critical evolutionary force that has shaped not just the survival of kidneys, but the whole trajectory of life. At its heart is a simple relationship, which may have begun with the origin of life itself – the reliance of virtually all cells on a peculiar kind of energetic charge, which Peter Mitchell named the chemiosmotic, or proton-motive, force. In each chapter of this book we’ve examined the consequences of the chemiosmotic force, but each chapter has concentrated on the larger implications of specific aspects. In the final few pages, I’ll try to tie all this together, to show how a handful of simple rules guided evolution in profound ways, from the origin of life, through the birth of complex cells and multicellular individuals, to sex, gender, ageing and death.