Cryopreservation of Complex Systems: The Missing Link in the Regenerative Medicine Supply Chain

Interestingness: 6

By Gregory M. Fahy, Brian Wowk, and Jun Wu. Rejuvenation Research. Summer 2006, 9(2): 279-291. doi:10.1089/rej.2006.9.279.

They pump the idea of using cryogenics to store pre-produced organs for use in transplants. They start with some interesting numbers regarding yearly US deaths that could be prevented if there were available organs:

• Heart  710,760
• Lung 122,009
• Kidney 37,251
• Liver 16,214

in total forming 36.2% of all yearly US deaths. They seem to be assigning all heart disease deaths as solvable by a heart transplant, etc. They also have a corresponding graph showing survival curves (ie age vs percentage alive) for status quo vs with those transplants, assuming no deaths during the transplants, with the transplant curve really diverging after 70 years of age with the transplant survival to status quo survival ratio of two at 80 and of 18 at 90. I don't know why they chose to show numbers that are so clearly not conservative.

They see cryopreservation as a necessary tool to manage the supply chain of organs manufactured through regenerative medicine to make mass organ availability viable. This is seen by them as the interim solution until in-situ recreation of the organ becomes a possibility.

They then describe a brief history of the cryopreservation of organs by straight freezing:

• 1950s - Freezing of guinea pig uteri to dry ice temperature (< -79C) and back. Contractile responses in vitro.
• 1973 - Freezing of dog intestines to liquid nitrogen temperature (< -196C). Long term survival of small parts of it.
• 2002 - Freezing of rat ovaries. Survival of some and offspring in one after transplantation back into rats. They say this is not a good test since the ovaries can regenerate even if completely screwed up.
• 2003 - Freezing of sheep ovaries. Vascular patency (unobstructed) retained in 3 of 11 grafts and follicle-stimulating hromone levels kept to normal limits. Second report claiming higher patency rates using directional freezing.

They claim straight-freezing of vital organs won't work because the vascular system is too easily damaged by freezing. This leads to methods that try to avoid freezing damage by avoiding ice formation.

In 1965, red blood cells and guinea pig uteri were lowered to -79C by lowering the freezing point of water and keeping it liquid all the way to the target temperature. Then in the 1980s, vitrification experiments started where the material is cooled into a glass without forming ice, starting with the vitrification of mouse embryos in 1985.

Experiments with vitrifications suggest that toxicity of cryoprotectant is due to reduced water availability for biomolecules. "Winner" of the cryoprotectants is M22 which seems to be used only below -22C with some other cryoprotectant (VMP) used to go from room temperature to -22C. Not much idea of what M22 consists of but it has a carrier solution that is meant to lower the toxicity combined with many anti-nucleating substances.

When ten rabbit kidneys were cooled to -22C, M22ised, deM22ised and rewarmed, then transplanted into rabbits, all rabbits survived (the non-cooled kidney is extracted in the experiments to make sure that the rabbits depend on the transplanted kidney), but there is more injury to the M22-ised kidneys than what is seen when just cooling them to -3C with VMP. Eight rabbit kidneys taken to -50C using M22 seem fine once rewarmed/deM22ised. Extra injury in M22-ised kidneys might be due to extra handling or extra time at cold temperature.

Newest experiment (2005) does the same rabbit kidney vitrification and transplantation on one rabbit with the rabbit surviving for 48 days after transplantation before it is killed. The cryoprotected kidney is cooled to below the glass transition temperature for M22 (-123C). Lots of damage to the kidney on this one, with hints that more even perfusion is needed.

Mitochondrial Microheteroplasmy and a Theory of Aging and Age-Related Disease

Interestingness: 6

By Rafal M. Smigrodzki and Shaharyar M. Khan. Rejuvenation Research. Fall 2005, 8(3): 172-198. doi:10.1089/rej.2005.8.172.

Long theoretical paper that tries to make a case for an accumulation of lots of rare mutations in mitchondrial DNA being a major cause of aging. By rare I mean that each individual mutation is rareish (1-2% of mitochondrial genomes), but between all of them it means that most of the mtDNA in a person is mutated in some way or other. The claim is that it is this accumulation of mtDNA mutations across the body plus the inherited component from the person's mother and, importantly, from the mutations accumulated in the mother's egg cell during her life prior to giving birth to the person in question, that causes the deterioration of the body.

The paper focuses a lot on Parkinson's and Alzheimer's disease, understandable since the first author is a neurologist. This made it harder for me to read as a "theory of aging" since I know almost nothing about either and Parkinson's is a relatively rare disease.

A lot of the arguments for why the primary cause should be in mtDNA or in DNA are similar to de Grey's arguments in de Grey's arguments in his version of the mitochondrial theory of aging so I will skip those. There are major differences in the details so I'll concentrate on those.

Low levels of lots of different mutations in the mtDNA (microheteroplasmy) are different from the uniform per-cell mutations of de Grey's. Its existence is much harder to detect but other studies supposedly show such microheteroplasmy exists in all tissue and increases with age, from about 45 mutations per million base pairs at birth to about 200 per million in old age (with a high starting level in the substantia nigra). Each mutation is usually only present in 1-2% of mtDNA across tissue but up to 10-20% in single cells.  By calculations, based on a low estimate of mutation rate, 90% of mitochondrial genomes have a mutation somewhere important. Based on a high estimate of mutation rate, there are on average 3 mutations per mitochondrial genome.  These numbers are very different from the usual numbers quoted and this is due to the trickiness of detecting microheteroplasmy (ie they are not detected at all by the usual methods of sequencing mtDNA) they say. Their referencing of reports of low levels of different types of mutations leading to high levels of mutations overall in sequencing of single neurons and glia are also at odds with the one mutational mtDNA taking over the cell's mitochondria that de Grey tends to reference.

The suggested mechanism of action is only in small part by causing insufficient amounts of ATP to be produced in the cell, but mainly by increasing reactive oxygen species (ROS) production to levels that damage the cell and/or drive it into senescence or apoptosis, with a possible feedback mechanism involved. They also postulate that even post-mitotic cells are regularly killed and replaced by cells derived from stem cells, and that accumulation of microheteroplasmy in the stem cells would lead to quicker reaching of the threshold amount of mutations in the derived cells needed for it to stop functioning properly (due to the above-mentioned mechanism). Overall though, the paper doesn't focus on the mechanism of action and does not rule out a different mechanism of action that would not go through ROS. In private communications with the first author, who gracefully supplied me with a copy of the paper and comments (Thanks Rafal), he mentioned he would downplay the effect of ROS on causing microheteroplasmy nowadays.

The paper has a long list of supporting evidence, but most of it seems quite tenuous. The strongest seems to be the case for Parkinson's disease correlating with mutations in a particular region of the mtDNA, which showed good predictive results in a small study. They mention correlations between Alzheimer's and complex IV dysfunction; cybrid models of both Parkinson's and Alzheimer's producing disease-specific responses (cybrids are hybrids of healthy cells without mitochondria fused with mitochondria from disease patients); faulty mitochondria in relatives and offspring of diabetes patients; oxidative stress and inheritance patterns in hypertension; glycolysis and cancer; metastasis in cancer as being a side-effect of old evolutionary reaction of the cell switching away from mitochondrial respiration as a source of power (due to mutated mtDNA).

The paper describes a particular type of mutation that they call focal microheteroplasmy, where a particular mutation becomes prevalent throughout a person due to it having originated during the mother's life prior to giving birth. While this mutation would be prevalent in the person's tissue, it would be completely different from other people in the family, even identical twins, who would have inherited a different half of the mitochondria. This mechanism is used as a partial explanation of the pattern of inheritance seen in certain diseases (eg Parkinson's and Alzheimer's).

The paper has an interesting list of corollaries/predictions of its hyptothesis:

• Focal microheteroplasmy in Parkinson's, Alzheimer's, diabetes and hypertension.
• Locations of the mutations in focal microheteroplasmy to be more detrimental than other locations of microheteroplasmy or of homoplasmic mutations.
• Accumulation rate of microheteroplasmy to be modulated by level of ROS.
• Menopause caused by microheteroplasmy, and trisomy 21 to protect the cell from microheteroplasmic-induced apoptosis
• Therapeutic interventions hammering secondary responses (eg amyloid in Alzheimer's) to be harmful
• Replacement of mitochondrial genomes to reverse many symptoms of aging

Overall, I'm not particularly fond of the mechanism of action being mainly down to ROS action. The predictions are quite good though, and if microheteroplasmy is as prevalent as stated then it should become quite clear shortly with the amount and variety of sequencing going on nowadays.

Selective Mitochondrial Autophagy, or Mitophagy, as a Targeted Defense Against Oxidative Stress, Mitochondrial Dysfunction, and Aging

Interestingness: 6

By Dr. John J. Lemasters. Rejuvenation Research. Spring 2005, 8(1): 3-5. doi:10.1089/rej.2005.8.3.

Short speculative piece about the importance of a mitochondrial outer membrane protein, Uth1p, in aging. Yeast cells with mutant versions of the gene have lower levels of mitochondrial autophagy when induced by rapamycin and starvation. If this means that the particular mitochondria that don't express Uth1p are not recycled, then it would give an "evolutionary" advantage for mitochondria to have mutant versions of this gene, as long as they are picked as often as others for mitochondrial replication. If this is the case, then other mutations in the same mitochondrion as ones with Uth1p mutations would proliferate throughout the cell.

As a downer, it seems there isn't (wasn't?) much information about the human analogue/s of the gene.

It's Never Too Late: Calorie Restriction is Effective in Older Mammals

Interestingness: 6

By Michael Rae, in Rejuvenation Research, May 2004, 7(1): 3-8. doi:10.1089/154916804323105026.

This is the first issue where the journal is actually called Rejuvenation Research.  It seemed to be a bit of a rebranding to try to disassociate from the general anti-aging crowd, and also when they switched from Michael Fossel to Aubrey de Grey as editor.  From the looks of this issue, it's a very positive change.

Most of this short review paper concerns itself with details of genetic profiles of calorie restricted (CR) vs all you want to eat (AD) mice, molecular differences shown, and potential problems with studies regarding CR and CR-mimetics.

There are two interesting bits for me. One is a graph showing proportion increase in lifespan in mice vs number of days under CR.  There is a reasonable straight line of best fit, with 45% increase in lifespan reached at around 1800 days on CR.

The other interesting bit is a table showing percentage of increased lifespan on mice when CR was started at weaning (1 month), 12 months (two of these), and 19 months.  Mean lifespans for these studies on the CR branch were 43, 37, 33 and 35 months respectively.  These represented 130, 119, 113 and 115 percent of control lifespans for each one.  Also, they lived 31, 18, 16 and 40 percent longer than controls from the point of starting CR.  Those are big numbers for percentage increase from starting point.

(That 19% longer on average vs 18% longer on average of remaining time for the second study seems dodgy to me.  It'd seem to imply the mice that did CR were already living 18% longer prior to start of CR). 

Noncorrelation Between Maximum Life Span and Antioxidant Enzyme Levels Among Homeotherms: Implications for Retarding Human Aging

Summary: de Grey thinks that we need superoxide dismutase in our mitochondrial intermembrane spaces

Interestingness: 6

Paper by Aubrey DNJ de Grey in the Journal of Anti-Aging Medicine, Volume 3, Issue 1, Spring 2000.

(((

This is another theory/speculation paper by de Grey. He's trying to explain why there is no correlation between life span and antioxydant enzyme levels in warm blooded animals. His explanation sticks to the oxidative damage is bad, m'kay, trend and tries to fit the model to the data.

He partitions the rate of damage created by oxidation into four:

• Specific metabolic rate (SMR): rate of consumption of oxygen per gram of body.
• Leakiness: Proportion of oxygen that becomes superoxide.
• Superoxide potency (SP): Proportion of superoxide converted into other oxide radicals (eg hydrogen peroxide) instead of being pacified by antioxidant enzymes.
• Oxidisability of the tissue (OT): How easily the relevant tissue gets oxidised.

He notes that under this scheme, levels of antioxidant vitamins affect the OT and not the SP, since they act mainly to stop the chain of oxidation. SP is the rate factor that is not correlated with lifespan that is being explained in this paper.

SMR in warm blooded animals (homeotherms) is mostly determined by body size, and lifespan does correlate with body size. Lifespan depends not only on size though but seems to be well correlated with lifespan restrictions imposed by external causes. There isn't much evolutionary pressure to raise the aging-based lifespan of the animal if it is likely to die from other causes (eg getting eaten). Animals of similar weight but different chances of dying due to external causes have different lifespans (eg birds vs mammals).

In a study in primates, superoxide dismutase (SOD) did correlate with lifespan if the SOD levels were divided by the SMR when doing the calculation. Catalase, glutathione peroxidase (GP) and glutathione (G) didn't though. A less dodgy comparison, in that it didn't need the division by SMR factor, was one between rats and pigeons. Pigeons live about 8 times longer than rats even though they weight about the same. SOD levels in the pigeon were slightly higher, catalase much lower, and G and GP levels varied depending on the tissue. Another study showed similar results when looking at the canary (very low mass, very high lifespan), with not even SOD showing higher levels.

In the same studies, they showed a correlation between leakiness and rate of aging, and maybe one between OT and rate of aging. Lending support to this second correlation, de Grey mentions that fatty acids saturation in the membranes of the mitochondria and levels of non-enzymatic anti-oxidants (eg vitamins C and E) are higher in longer lived animals and these lower OT. This supposedly leaves SP as the only one out of the four factors that doesn't correlate in the predicted way with longevity.

de Grey's hypothesis to explain this is that there is no easy way for evolution to lower the SP because there are no SOD enzymes in the mitochondrial intermembrane space (MIMS) to mop up the superoxides. The selection for longer lifespan instead pushes the concentrations of non-enzymatic antioxidants (vitamins C and E) up all over the cell to get levels up in the MIMS, and the leakiness of the MIMS down which is the same mechanism that controls leakiness elsewhere in the chain. This then means that the concentrations of antioxidant enzymes in the non-MIMS regions become too high for the resulting lower radicals due to the improved leakiness and non-enzymatic antioxidant profiles, and these enzymatic antioxidant levels drift down to save resources until they match the levels that would lead to the same rate of damage as the other parts of the oxidation chain.

Since that paragraph contained the whole hypothesis I will write it again, but in expanded form. Homeotherms supposedly don't have any SODs in their MIMS but we do produce superoxides there (the evidence for that second part is probably not great). The damage caused by this, somehow (more on this later), limits our lifespan. For intelligent or otherwise flighty animals, where the external causes of dying are lower, there is a selective pressure, apparently, to raise our lifespan due to aging to match the lower external causes. Since it seems to be troublesome to introduce a SOD into our MIMS (and this supposed trouble to evolve a MIMS-SOD is the bit that to me seems weakest out of the chain of reasoning), homeotherms instead reduce the leakiness of the ATP-making mechanism, the leakiness factor, and raise the levels of non-enzymatic anti-oxidants, lowering the OT factor, to lower the total rate of aging. Now, lowering the leakiness of the process lowered the production of oxidants everywhere, not just in the MIMS, and raising the level of non-enzymatic anti-oxidants did the same everywhere, not just in the MIMS, so now, if we kept the same level of enzymatic anti-oxidants as before these last two improvements, the level of oxidants everywhere non-MIMS becomes too low for the available enzymatic anti-oxidants. By too low, he means that the bottleneck will be the MIMS oxidants, and everywhere else the oxidant damage will always be too low to matter. Since now the organism can get away with lowering the enzymatic oxidant levels in the non-MIMS sections, it does so, since it saves energy doing so.

That this non-correlation between enzymatic antioxidant levels and lifespan does not occur in flies and worms, (ie, in those species, the correlation does exist and is positive), means that the lifespan-limiting mechanism in flies and worms is different from homeotherms. de Grey suggests that this mechanism is the mutation of mitochondrial DNA (mtDNA) which tends to be attached to the inner surface of the inner membrane of the mitochondria. The mtDNA would somehow be damaged by the higher unquenched superoxide concentration across the inner membrane, in the MIMS. Old mammals have been shown to have high levels of mtDNA mutations, while this doesn't happen in flies and worms, maybe because they do not live long enough for the process of mtDNA amplification to take place. He's trying to tie it all back to his other paper (http://readingrejuvenationresearch.blogspot.com/2010/01/proposed-refinement-of-mitochondrial.html)

The suggested methods for testing the hypothesis: retarget MnSOD and CuZnSOD to the MIMS and check that they are useless there. If they are not useless, then it should have been easy to evolve those. Afterwards, retarget E Coli's iron-dependent SOD to the MIMS of mice, as has supposedly been done before in yeast, and see if that affects lifespan. That last check doesn't make sense to me. If the enzymatic anti-oxidants in the non-MIMS sections have drifted down until they are causing as much trouble as the MIMS oxidation, then lowering MIMS oxidation damage shouldn't affect the lifespan of the beasts. Doing this while raising enzymatic anti-oxidants throughout the cell might though.

In conclusion, another very interesting chain of causation hypothesis, but probably too long to have much of a chance of being correct.

)))


Abstract follows:

A series of studies over many years has conclusively disproved the hypothesis that longevity in warm-blooded animals (homeotherms) correlates with high levels of antioxidant enzymes: in fact, these variables generally exhibit a strong negative cross-species correlation. In flies and nematodes, however, substantial extension of maximum life span has resulted from genetic manipulations that increase antioxidant enzyme levels; these manipulations have always been associated with increased resistance to oxidative challenge, indicating that the life span extension is directly caused by the raised antioxidant capacity. This stark contrast merits careful analysis because it casts doubt on the value of short-lived invertebrates as models for the investigation of mammalian aging. Here is it shown that these results imply the existence, in homeotherms but not in flies or worms, of life span-limiting pathways of oxidative damage that are impervious to enzymatic antioxidants. This is shown to lend weight to the currently controversial theory that somatic mitochondrial DNA mutations contribute significantly to determining the rate of aging of homeotherms, and thereby suggests a feasible intervention to retard human aging.

Is Telomere Shortening Related to Progeria?

Summary: Telomere shortening probably doesn't cause Hutchinson-Gilford progeria

Interestingness: 6

Paper by W Ted Brown in the Journal of Anti-Aging Medicine, Volume 2, Issue 2, Summer 1999.


(((
This is a short note speculating on whether progeria is caused by telomere shortening. The author says unlikely. Hutchinson-Gilford progeria is a fucking rare disease (1 in 8 million) of the type that pop up through a dominant spontaneous DNA mutation. Progerias are diseases that look like accelerated aging. This one starts being noticeable in toddlers between one and two years old, then they start looking old very quickly, going bald and losing subcutaneous fat, and have an expected lifespan of 13 years. 80% of them die of heart attacks and congestive heart failure, but they don't seem to get cancer, cataracts, osteoporosis or Alzheimer's like regular old people.

Fibroblast cultures extracted from progeria patients have an almost normal lifespan, but one paper reported shorter telomeres in them. Studies from Werner's syndrome, a different progeria that hits during early adulthood, give mixed results for shorter telomeres, but maybe some indication of faster telomere shortening.

Mice with telomerase knocked out don't show too many problems and in one study, could reproduce for at least six generations. By the sixth generation, their telomeres were much shorter and there were a lot of chromosome fusions. Other studies on these telomerase knockouts showed slightly lower lifespan, lower wound healing capacity, and more cancer. From this, he says it seems unlikely that telomere shortening would cause progeria. From what I remember, though, mice have way longer telomeres than humans to begin with, which would hide the effect a bit, but he didn't discuss that

)))

Aging: Minimizing Free Radical Damage

Summary: The founder of the free radical theory of aging summarising the results that back the theory, some nice graphs, and other interesting bits of speculation.

Interestingness: 6

Paper by Denham Harman, MD, PhD, in the Journal of Anti-Aging Medicine, Volume 2, Issue 1, Spring 1999.

(((This is a summary of the current state of the free radical theory of aging (FRTA) by the guy that is introduced as the father of the FRTA. I didn't like the way it was written. I'm going to skip big chunks of it)))

(((The paper starts with a series of mortality curves over age across time for women in Sweden from the 1750s to 1992. These are cool, even if I've seen them before. They show the mortality following Gompertz function with mortality going up exponentially after around age 50 with a doubling time of about 7 years. The slope of this exponential is the same in all curves. While mortality is much lower across all ages as we get closer to the present, the line goes exponential at a younger and younger age, so that the difference in mortality at ages 70 onwards is not that big across history. So, for example, the curve for the 1900s and 1920s seem to hit the exponential proper only at age 60, while the curve for 1992 seems to be on the exponential from age 40. The left hand side of the curves, that is, the bits before we hit the exponential growth, have declined massively across history. The text mentions that in that 1992 curve, only 1.1% of all females in Sweden die before age 28 (the date at which he puts the exponential rise starting) )))


(((It continues with a couple of life expectancy graphs from the 1950s to the present for male and females in Sweden, Switzerland, the USA and Japan. The first three going up by 1-2 years per decade and Japan by 3 years per decade, from a lower base. I don't really understand what these graphs or the previous mortality curves have to do with the main theme of the article, but I like them anyway)))

The free radical theory of aging (FRTA) says that all aging and death in all living things is based on the initiation of free radical reactions, the rate of which is determined by genetics and environment. This theory was later extended (((modified?))) to say that in mitochondria-containing living things, it is the rate of initiation of free radical reactions (FRR) in the mitochondria that determines their lifespan. FRRs can be classified into initiation, a propagation chain, and termination. An antioxidant usually refers to a compound that breaks the propagation chain, or, in general, any substance that delays or inhibits oxidation in low concentrations.

The major sources of radical reactions are:

• Respiratory chain
• Phagocytosis
• Prostaglandin synthesis
• Cytochrome P-450 system
• Nonenzymatic reactions of O2
• Ionising radiation

Defenses against damage caused by FRR are:

• Antioxidants. eg: tocopherols, carotenes
• Heme-containing peroxidases. eg: catalase
• Glutathione peroxidase
• Superoxide dismutases (SOD)
• DNA repair mechanisms

By the FRTA, slowing down FRRs would increase longevity. Studies backing this up include:

• Overexpression of superoxide dismutase and catalase in fruit flies extended life span by a third.
• Longer-lived strains of fruit flies, flatworms and bread mold have higher antioxidant enzyme activity than short-lived strains
• Addition of 2-mercaptoethylamine (2-MEA), an antioxidant, to food increased average lifespan of LAF1 mice by 29.2% (((no idea what the characteristics of LAF1 mice are)))
• 2-MEA addition to food of mice mothers before mating increased lifespan of their offsprings by 15% and 8% to male and female offsprings respectively

Decreasing initiation rates of endogenous FRRs would also lead to increased longevity. The rate can be reduced by caloric reduction, compounds that compete with O2 for access to electron-rich areas of the mitochondria, compounds that bind to the respiratory chain and stop the reaction with O2, and genetic regulation of mitochondrial superoxide creation. Cutting caloric intake of rats by 40% increased average lifespan by 40% and maximal life span by 49% (((Those numbers are higher than I'm usually accustomed to))). The study also suggests a lower rate of aging for rats under caloric restriction (((ie a lower gradient on the semilog plot of age vs mortality))) (((I think the suggested link is lower amount of products to oxidise => lower total load of FRR in mitochondria))).

Only study showing antioxidant to extend maximal lifespan of mice is 2-MEA, added at 0.25% w/w to the diet of BC3F mice extended mean and maximal lifespan by 13% and 12% respectively. The study hasn't been replicated. The reason that most antioxidants fail to extend lifepan is that they have toxic effects on mitochondria at lower concentrations than those needed to slow down FRRs significantly.

The paper continues by listing the possible effects of the FRTA on specific diseases. They are:

• Cancer, listing epidemiological studies suggesting vitamin C and fruits and vegetables having lower incidence
• Atherosclerosis, caused by lesions that would result in higher localised concentrations of oxidation products, and oxidation of polyunsaturatid lipids, and mentioning a study of vitamin E supplementation showing a decrease of 40% in coronary artery disease (((never heard of that one. will have to look it up)))
• Hypertension, mentioning a study of SOD targeted to endothelium cells lowering blood pressure in spontaneously hypertensive rats, but not in normal rats
• Alzheimer's disease, listing mutations in mtDNA, mutations in amyloid precursor protein (APP), and increases in levels of APP and SOD in Down's syndrome (((I don't get how the last two are meant to relate to FRTA)))
• Immune deficiency, saying some antioxidants increase immune responses
• Autoimmunity, with ethoxyquin fed to a mice used for studying autoimmune disease (NZB) increasing lifespan by 32%

The gender mortality gap is also supposedly explained by the FRTA via two different effects: one is the lower stores of iron in women prior to menopause leading to less FRRs catalysed by iron, and the second (((something I don't even understand enough to describe))).

The paper finishes by claiming that a large part of the increase in lifespan in the USA since the 1960s could be attributable to the widespread use of multi-vitamins by the population (((yeah riiiiiight))).


Abstract follows:

Aging is the accumulation of changes that increase the risk of death. The major contributors after age 28 years are the endogenous chemical reactions that, collectively, produce aging changes that exponentially increase the chances for disease and death with age. These reactions constitute the "inborn aging process." This process is the major risk factor for disease and death of the 98% to 99% of cohorts still alive at age 28 in developed countries, where living conditions are now near optimum.

The Free Radical Theory of Aging (FRTA) and, simultaneously, the discovery of the ubiquitous, important involvement of endogenous free radical reactions in the metabolism of biologic systems, arose in 1954 from a consideration of aging phenomena from the premise that a single common process, modifiable by genetic and environmental factors, was responsible for the aging and death of all living things. The FRTA postulates that the single common process is the initiation of free radical reactions. These reactions, however initiated, could be responsible for the progressive deterioration of biologic systems with time because of their inherent ability to produce random change. The theory was extended in 1972 with the suggestion that the life span was largely determined by the rate of free radical damage to the mitochondria.

The FRTA suggests the possibility that measures to decrease the rate of initiation and/or the chain length of free radical reactions may, at least in some cases, decrease the rate of reactions that produce aging changes without significantly depressing those involved in maintenance and function. Many studies support this possibility.

Applications of the FRTA have been fruitful. For example, it is a useful guide to efforts to increase the life span, and it provides plausible explanations for the aging phenomenon (e.g., the association of disease with age as well as insight into pathogenesis; the gender gap; the association between events in early life and late onset disease; and the shortening of telomeres with cell division). Further, it is reasonable to expect on the basis of animal and epidemiologic studies that the increasing population-wide use of antioxidant supplements and ingestion of foods high in antioxidant capacity over the past 40 years have helped to increase the functional life span of the population by contributing significantly to the decline in "free radical" diseases, to increases in the fraction of elderly, and to the decline in chronic disability in this group.

A Mechanism Proposed to Explain the Rise in Oxidative Stress During Aging

Summary: Longish speculative chain on how cells dominated by mutant mitochondria, even though they are rare, can cause system wide oxidation damage. The speculation sounds speculative.

Interestingness factor: 6ish

Paper by Aubrey de Grey, published in Journal of Anti-Aging Medicine, Volume 1, Issue 1, Spring 1998. (((can be gotten from http://www.sens.org/files/sens/AdGpubs.htm)))

(((A long theoretical piece. I'm not going to do it justice. Reading about glycolysis and the Krebs cycle helps)))

The previous blog reviewed the article that forms the basis for this paper. Assuming that mutant mitochondria take over individual cells, how can they affect the rest of the body, if, as this paper points out, they seem to only make up about one percent of tissue cells?

The hypothesis starts with an explanation of how these cells survive at all. It uses data gathered from experiments with cells that lack mitochondrial DNA altogether (p0 cells), that can survive if supplemented with pyruvate or a whole bunch of other molecules.

The problem to solve for them, aside from the lower amounts of energy available, is how to restore levels of NAD+ that get converted to NADH during glycolysis. The proposed solution is that they do this by reduction of extracellular molecules through an enzyme that sits on the membrane called plasma membrane oxidoreductase (PMOR) that exist in every cell. That is, they do this by exporting electrons. Evidence presented is that succinate dehydrogenase is upregulated in these p0 cells, and that since it is part of the Krebs cycle that consumes pyruvate, it shows that the main alternative method of exporting those electrons (by reducing pyruvate to lactate with NADH and then exporting the lactate) is probably not being used. (((Which doesn't show me how some other third method is not what is really going on)))

Those exported electrons primarily go to vitamin C in extracellular fluid, but once you run out of that, they'd go to oxygen, creating superoxide radicals. Most of this would be cleaned up by superoxide dismutase, but some would escape and react with the iron in haemin (((non-protein bit of haemoglobin))) (other iron options are well protected) which would then oxidase LDL particles. This last part, the oxidation of LDL particles by haemin, seems to have some evidence behind it.

The oxidation of some LDL would raise the intake of somewhat oxidised LDL by all cells in the body, which would raise the amount of oxidation damage that all cells have to deal with. Since this mechanism would be going on constantly, ie those mutant mtDNA cells would be constantly spewing electrons, quite a lot of oxidised LDL would be created.

The paper then moves onto methods of testing the suggested chain of events:

• Seeing if cells that do not have a functioning electron transport chain (by assessing cytochrome c oxidase activity) have high PMOR activity.
• Seeing if there's high levels of superoxide near cells with busted mitochondria
• Checking if LDL is highly oxidised near mutant cells
• Checking if oxidised LDL particles stress normal cells anti-oxidant system.

And then onto methods for checking that it affects aging:

• Restoring the function of the mutant mitochondria by importing the proteins encoded by the mitochondrial DNA and seeing what happens (((hard project)))
• Targeting those zombie cells controlled by mutant mitochondria and destroying them, then seeing what happens. (((I like it)))

(((Conclusion: The chain of events suggested here sounds much more dubious to me than the one suggested in the previous article. The electrons from NADH might be used some other way inside the cells, or the electrons might be quenched in some benign way outside the cell, or oxidation of LDL might not have any major effect on aging (outside of the effects on cardiovascular disease). The tests seem simple enough though, and they'll pop up regardless, if they haven't already. It would be good if this was correct since killing the mutant cells doesn't sound insanely hard to me. Easier than curing cancer since these cells don't reproduce, as determined by the method that they come into existence)))

(((This is the last paper that I'll write about in the first issue. The rest consists of some futurist speculation, a meeting report and literary review. While interesting, they are already in summary form)))

Abstract follows:

Most phenotypes of aging in vertebrates may be caused by a progressive decline in the ability of antioxidant defences to maintain cellular and systemic homeostasis. This is due both to a diminished efficacy of those defences and to an enhanced level of pro-oxidant toxicity; the imbalance between the two has been termed oxidative stress. However, the cause of this increasing imbalance remains obscure. This article proposes a mechanism by which spontaneously mutant mitochondrial DNA (mtDNA), despite being present only in very small quantities in the body, may be the main generator of oxidative stress. Mutant mtDNA is distributed very unevenly within a tissue: some cells apparently contain no wild-type mtDNA whatever. Those cells must rely on glycolysis for ATP production; furthermore, they require a system to stabilize their NAD+/NADH ratio. This can only be achieved by an efflux of electrons from the cell, most probably mediated by the plasma membrane oxidoreductase (PMOR). It is proposed that the required rate of electron efflux from these anaerobic cells exceeds the local electron-accepting capacity of "safe" acceptors in plasma such as dehydroascorbate, with the result that reactive species, such as Superoxide, are formed. This leads to increased oxidation of lipids in the plasma, notably of low-density lipoprotein (LDL) particles, which are subsequently imported into mitochondrially healthy cells. This oxidized lipoprotein must be destroyed by the recipient cells' antioxidant defences. That task diverts the cell from the degradation of pro-oxidants that it is itself generating; thus, it imposes oxidative stress on the cell. As the number of anaerobic cells in the body rises, so does oxidative stress in all cells. The consistency of this hypothesis with known facts is discussed, and technically feasible tests are suggested both of the proposed mechanism and of its overall contribution to mammalian aging, including plausible interventions to retard the process.