The Mitochondrial Free Radical Theory of Aging - Supernova: Pliki

The Search for How So Few Anaerobic Cells Cause So Much Oxidative Stress
reason—lost the ability to make use of oxygen would rapidly die. Yet, as noted in Sections
6.4 and 6.6, it seems that such cells in fact accumulate. How? Well, the alternative possibility
is that they do not really survive—that they struggle on for a short while and then die. But if
that is indeed what happens, we’re back to a turnover problem (see Section 5.6): the only
reason why we see more of them in older individuals must be because they are occurring
more often and/or being killed off more sluggishly, and this itself would need an explanation
in terms of a prior cause of oxidative stress.
But the alternative of indefinite survival of these cells has its own problem: it seems, on
the face of it, necessarily to require them to exchange some metabolite with still-aerobic
cells, via the extracellular medium. But then, we would expect to find this metabolite in
plasma, at levels that increase with age. No substance has been found which fits this criterion.
For example, the most obvious candidate—a simple exchange of lactate, allowing the
anaerobic cell to maintain glycolysis without any ancillary process—appears to be ruled out
because plasma lactate was not observed to rise with age. 10
However, a breakthrough was made a decade ago which showed that this option—that
cells with no OXPHOS functionality nevertheless survive indefinitely—was still a possibility
after all.
I mentioned in Section 6.3 that various fungi have, since the 1950s, been observed to
undergo spontaneous “suppressive petite” mtDNA mutations which take over their cells.
The cells that suffer this phenomenon are totally incapable of aerobic respiration, relying
purely on glycolysis, and they grow much more slowly than other cells. But they do grow.
This is because the fungi in question are facultative aerobes, which can survive without
oxygen (that is, without OXPHOS) if they have to. Human cells in culture, by contrast, are
obligate aerobes and die rapidly when divested of OXPHOS function, even in the presence
of unlimited glucose.
In 1989, however, King and Attardi succeeded in creating a viable human tissue culture
cell line which had no mitochondrial DNA. 11 Such cultures are termed “ρ 0 ” lines. This was
a major advance, because until then the only cells that were able to grow without fully
functioning mtDNA were ones (like yeast) which can grow without oxygen anyway. It was
fairly easy to remove the mtDNA—low concentrations of ethidium bromide, a chemical
that inhibits mtDNA replication but not nuclear DNA replication, 12 achieved this without
killing the cells—but in order to keep the cells alive and dividing indefinitely they had to
find the right nutrients, and, indeed, glucose on its own was not adequate. Two further
nutrients turned out to be absolutely vital. One was uridine, which is an intermediate in
nucleotide synthesis and was needed because one enzyme involved in its synthesis uses the
respiratory chain. 13 The other was the one of most relevance here, though: it was pyruvate.
Why on earth pyruvate? The cells were being given plenty of glucose, and were clearly
breaking it down to make ATP; pyruvate is the product of this breakdown, so obviously it
was in plentiful supply already. Yet, with glucose but no pyruvate, the cells rapidly died.
The answer lies in the cofactors. When glucose is broken down to make two molecules
of pyruvate, simultaneously—and unavoidably—two molecules of NAD + are turned into
NADH (see Fig. 2.3). There is no escaping this: pyruvate has two fewer than half the electrons
in glucose, so there are four electrons to lose. However, pyruvate can easily be turned
into lactate; splendidly, lactate has two electrons more than pyruvate, so the NADH can be
restored to NAD + . The net conversion of NAD + to NADH that results from the conversion
of glucose to lactate is thus zero. This is convenient in the short term, since it means that
cells can briefly engage (during strenuous activity, for example) in faster energy expenditure
than is allowed by the oxygen supply: they can burn glucose and make lactate at a rate not
limited by the supply of oxygen. Lactate is toxic though, so after a short time we have to
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