Downregulation of Glutamine (GLN) Synthetase Activity in Response to GLN: Relevance to Research on GLN as an Energy Substrate and "ATP-Sparing Agent"

In this article [Mignon et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17947599?dopt=Abstract)], Mignon et al. (2007) found that glutamine (GLN) supplementation only produced statistically-significant reductions in the activity of glutamine synthetase (GS), in the skeletal muscles, in the fed state in aged rats and in the fasted state in adult rats. The GLN-induced decreases in GS activity in the other "states" (fasted state in aged rats and fed state in adult rats) were not statistically-significant. It's interesting that the tissue concentrations, which are going to be mainly intracellular, of GLN and glutamate and plasma concentrations of GLN and glutamate did not increase in response to supplementation. Those findings, when viewed in alongside the reductions in GS activity, are consistent with my sense of the way GLN supplementation is likely to exert its supposed therapeutic effects [see here for my bare-bones paper on GLN: (http://hardcorephysiologyfun.blogspot.com/2009/08/some-more-old-papers-of-mine.html)], as discussed below. Mignon et al. (2007) cited research that had shown that hypermetabolic, or "catabolic" states, such as can occur after surgeries or other causes of physiological stress, have generally been associated with an upregulation of GS activity, and researchers have typically attributed those increases in GS activity to glucocorticoid-mediated increases in the mRNA expression of GS or to other factors, etc.

That research by Mignon et al. (2007) is relevant to the use of GLN as an energy substrate, in general, and to its use as an "adjunctive" energy substrate in the treatment of depression, etc. There's only one article on the use of GLN as an adjunctive antidepressant [Cocchi, 1976: (http://hardcorephysiologyfun.blogspot.com/2009/03/gabaergic-effect-of-l-glutamine-in-rats.html)], and its efficacy has obviously not been proven and will never be proven. But that article by Cocchi (1976) is remarkable in the sense that the author's observations are generally consistent with the kinds of effects that one would expect to see, based on all the research that has been done, in response to GLN. The author also noted that the therapeutic window was relatively narrow, and, in my experience, it's extremely narrow and changes in response to changes in exercise intensity and to changes in factors that affect serum calcium (such as vitamin D). All I can do is relate my sense of things, and I don't have a good explanation for the reason the range of therapeutic dosages would be so small. I mean that tiny increases in the dosage can either produce beneficial effects, in terms of the effects that one would ideally expect from an energy substrate, under some conditions, or can cause effects that seem to be consistent with the GABAergic effects that Wang et al. (2007) described [see that past posting for my discussion of this: Wang et al., 2007: (http://www.fasebj.org/cgi/reprint/21/4/1227)(http://www.ncbi.nlm.nih.gov/pubmed/17218538?dopt=Abstract)].

The finding that exogenous GLN can decrease GS activity without increasing the steady-state intracellular GLN concentrations in skeletal muscle myocytes (and satellite cells, etc.) is significant in relation to an understanding of GLN metabolism in general, and the finding can be explained by the fact that exogenous GLN can increase the 26S-proteasomal degradation of the GS enzyme protein [Labow et al., 2001, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/08/some-more-old-papers-of-mine.html)]. That's really important, but there's some sort of resistance to the fact that GLN is likely, as it is, in my opinion, to exert many of its effects by virtue of its capacity to serve as an energy substrate. There are many articles that have shown this, and I'm not going to collect all of them right now [the protection by GLN against damage due to ischemia is basically a result of its capacity to be converted into 2-oxoglutarate and undergo oxidation in the TCA cycle, and here are some of those articles showing protection against ischemic damage: (http://scholar.google.com/scholar?q=glutamine+ischemia&hl=en)]. There's at least one article showing that it improves cardiac function acutely, in humans with heart failure or heart disease [here it is: Khogali et al., 2002: (http://www.ncbi.nlm.nih.gov/pubmed/11844641)].

The key point, however, is that GS activity consumes enormous amounts of ATP, and very few tissues in the body are characterized by a net formation of GLN. There are all of these articles discussing the fact that the GLN-glutamate-GABA cycle accounts for 70-80 percent of the ATP consumption in the brain, and a lot of articles emphasize the fact that astrocyte-derived GLN is utilized as a major energy substrate for neurons. But the downregulation of GS activity by exogenous GLN is likely to not be accompanied by major increases in either the steady-state extracellular or intracellular GLN or glutamate concentrations, and, following a brain injury, there might not even be any post-infusion, detectable increase in the extracellular-fluid GLN concentrations in the brain [the CNS "parenchymal" interstitial fluid (ISF) concentrations]. This phenomenon has been shown in the liver and in cultured cells, also [see Yudkoff et al., 1988, and Qu et al., 2001, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/05/problems-with-glutamine-research.html)], and I've cited all the research in past postings. The turnover is so rapid and so massive that an infusion of even multi-gram amounts, in the context of the 23 to 60-fold increases in the rate of oxidation of GLN carbons in the TCA cycle that occur in the brain, following ischemia [see here: (http://hardcorephysiologyfun.blogspot.com/2009/05/oxidation-of-glutamate-derived-2.html); Pascual et al., 1998: (http://stroke.ahajournals.org/cgi/content/full/strokeaha;29/5/1048)(http://www.ncbi.nlm.nih.gov/pubmed/9596256)], could easily fail to elevate ISF GLN in the brains of people who have traumatic brain injuries. But the downregulation of GS activity by GLN could, nonetheless, spare significant amounts of ATP, and, of course, ATP depletion is going to occur sooner or later after a brain injury. One can sometimes show no ATP depletion for a little while after an injury, but that's probably because structural damage to the mitochondria takes a couple of days to occur. Another reason that the GLN-mediated decreases in ATP consumption by GS activity would be desirable, in my opinion, is that glutaminase can, especially under those conditions in which the oxidation of GLN carbons is drastically augmented (i.e. after a brain injury or even, arguably, under more mild conditions of deranged energy metabolism), escape feedback inhibition by intramitochondrial glutamate. Essentially, glutamate formed by the glutaminase-mediated deamidation of GLN (in the mitochondria) is likely to be oxidized or otherwise utilized with exceptional rapidity, and that means that the pool of glutamate that is available to exert feedback inhibition of glutaminase activity [see here for discussion: (http://hardcorephysiologyfun.blogspot.com/2009/05/oxidation-of-glutamate-derived-2.html); Brand and Chappell, 1974: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1167992&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/4375961)] is going to be even more limited than it usually is. That change in the normal allosteric regulation of glutaminase could create an ATP-consuming futile cycle, for all practical purposes, in tissues following ischemia, and GLN could be one approach to breaking that futile cycle. Anyway, the point is that GLN could reduce ATP consumption in skeletal muscles ("spare" ATP) or in the brain [it does cross the blood-brain and blood-CSF barriers, and that's apparent and is discussed in articles cited here: (http://hardcorephysiologyfun.blogspot.com/2009/08/some-more-old-papers-of-mine.html)] without necessarily producing drastic or even any changes in the tissue or plasma or ISF GLN concentrations, particularly following ischemia or hypoxia or other physiological stressors that can, as found by Pascual et al. (1998), cited above, increase the percentage (and rate) of the intracellular GLN-derived glutamate pool that is oxidized, upon its metabolism into 2-oxoglutarate, in the TCA cycle. The rates of GLN synthesis, by ATP-consuming GS, and degradation are very high in many tissues, and that's one reason that so few cell groups display an overall, net output of GLN. At very high or otherwise excessive GLN intakes, the adverse effects of the extra ammonia could conceivably outweigh the benefits associated with the supposed ATP-sparing effects. GLN could also interfere with the transport of citrulline or other amino acids or intermediates, as discussed in past postings.

Incidentally, other researchers [Young et al., 1993: (http://www.ncbi.nlm.nih.gov/pubmed/8289407); Morlion et al., 1998: (http://www.pubmedcentral.nih.gov.floyd.lib.umn.edu/picrender.fcgi?artid=1191250&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/9488531)] have reported that people who had been treated with intravenous L-alanyl-L-glutamine (the stable dipeptide "form" of glutamine that can be stored in i.v. solutions in the long term) had noted improvements in "mood" or "well being." It's easy to dismiss things like that, but it's possible to easily dismiss things to the detriment of...oneself. "It's not necessarily *good* to be dismissive of *things*." That's the end of this posting.

Adenosine vs. Ribose vs. AICAriboside for the Restoration of Adenosine Nucleotides in the Heart Following Ischemia

In this article [Mauser et al., 1985: (http://circres.ahajournals.org/cgi/reprint/56/2/220.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/3918804)], Mauser et al. (1985) show that the intraarterial infusion of adenosine produced a 90-fold increase in the rate of adenosine nucleotide resynthesis (mostly by purine salvage pathways), following cardiac ischemia in dogs, and infusions of equimolar dosages of ribose or AICAriboside produced only between 5-fold and 9-fold increases in the rate of adenosine nucleotide formation, by either the salvage or de novo pathways. Only adenosine significantly restored ATP levels, following ischemia. All of the compounds were infused intraarterially, into the left coronary arteries of the dogs.

This article shows, in my opinion, that adenosine is far superior to ribose as an approach to restoring adenosine nucleotide levels following their depletion, and the research casts doubt on the idea, as suggested by the authors of some articles, that the entries of the carbons of ribose, derived from exogenous purine nucleotides, into the nonoxidative pentose cycle and into glycolytic pathways make a substantial contribution to the purine-mediated protection of cultured cells, such as astrocytes, against death due to glucose deprivation or other conditions. In most of those articles, the only evidence that ribose mediates the protective effects is that purine nucleoside phosphorylase (PNP) inhibitors sometimes block the protective effects of exogenous purines. But, as discussed by other authors, that doesn't mean that the use of ribose as a glycolytic substrate mediates the protective effects of purines or that ribose can substitute for the preformed nucleotides. It may just mean that ribose-1-phosphate has to be derived from nucleoside phosphorolysis to maintain purine salvage and that, paradoxically, more purine nucleobases are lost when ribose is "locked" in nucleosides and nucleotides than are lost when some turnover of nucleoside-derived ribose is allowed to occur, via the PNP-mediated formation of ribose-1-phosphate and purine bases. Also, the inhibition of nucleoside phosphorolysis prevents the formation of uric acid from the exogenous purines, and some authors have suggested that uric acid-induced peroxynitrite scavenging may partially mediate the protective effects of purines on cultured cells. There's a lot of research showing that uric acid can maintain mitochondrial functioning, in cells in the liver or in other cells, by preventing the inactivation of respiratory-chain enzymes by peroxynitrite, etc. Additionally, adenosine is normally kept at a very low concentration intracellularly, and massive amounts of adenosine have to be supplied, normally, to produce adenosine-mediated toxic effects and S-adenosylhomocysteine accumulation, etc., in cells [usually 1 mM (1000 uM) or higher of extracellular adenosine is required, a concentration that is supraphysiological] [Adair, 2005: (http://ajpregu.physiology.org/cgi/content/full/289/2/R283)(http://www.ncbi.nlm.nih.gov/pubmed/16014444)]. If adenosine were to accumulate as a result of PNP inhibition, that accumulation could produce toxic effects. But adenosine is normally metabolized extremely rapidly.

The rate of de novo purine biosynthesis is extremely slow, and this is one reason, as Mauser et al. (1985) discussed, that AICAr did not produce very significant restorative effects on adenosine nucleotide levels. Ribose did not even appear to contribute much to purine salvage, in my opinion, in comparison to the effects of adenosine. Even if the effects of ribose depended on its metabolism into glycolytic intermediates, the presence of the preformed purine nucleotides, such as can be derived from exogenous adenosine, appear to be crucial and to be a limiting factor in the rate of ATP resynthesis and nucleotide replenishment following ischemia. There are other articles that provide similar data.

In the case of pyrimidines, the research showing that uridine phosphorylase (UP) inhibition can abolish the cytoprotective effects of exogenous uridine, as in astrocytes, tends to not take into account the role that UP is thought to play in the salvage of uracil in rodents and cultured astrocytes, etc. The traditional view is that pyrimidine bases are not salvaged and that pyrimidine salvage occurs only at the level of the whole nucleoside, meaning that the main salvage pathway for uridine, for example, is its phosphorylation to UMP by uridine kinase. Some of these articles discuss the fact that, in the brains of rodents, UP appears to play an "anabolic," rather than catabolic, role and to be required for pyrimidine salvage [Mascia et al., 1999, etc.: (http://scholar.google.com/scholar?q=%22uridine+phosphorylase%22+salvage&hl=en&lr=)]. Also, inhibition UP may indirectly inhibit purine salvage, given that UP inhibition would prevent uridine from serving as a source of ribose-1-phosphate. This could decrease the formation of PRPP from uridine-derived ribose-1-phosphate, thereby increasing the loss of purines and compromising both ATP formation and the ATP-dependent salvage of uridine, etc. The depletion of purines has been shown to lead to secondary depletion of pyrimidines, even in the context of the fructose-induced depletion of ATP from the liver. Fructose has been shown to transiently elevate plasma uridine levels, and that's very much a pathological effect, in my opinion. Some people seem to think that the fructose-induced elevation of plasma uric acid levels is "good" or desirable, given that uric acid scavenges peroxynitrite. But the elevations in uric acid levels, following fructose ingestion or infusion, are mainly the result of pronounced ATP depletion in the liver, in my opinion (and as shown by countless articles). That's not desirable. In any case, the articles in these areas are interesting.

Folate and UVB Papers in Pdf Format

Here are my old papers, with the diagrams included, in pdf format. I now think that reduced folates, such as L-leucovorin (L-folinic acid) and L-methylfolate, are far superior to folic acid, for many reasons, but the concepts discussed in the folic acid paper are still "valid." Here's the paper on folate metabolism in relation to purine and pyrimidine metabolism (http://www.mediafire.com/?zmfojwc4am2), and here's the paper on the effects of cutaneous UVB/UVA exposure on sensory neurons and projection neurons/wide-dynamic-range neurons in the dorsal horn and caudal trigeminal nucleus, etc. (http://www.mediafire.com/?410jmzmrjjm).

Uridine-Induced Maintenance of Glycogen and Total Adenosine Nucleotide Concentrations During Hypoxia: Apparent Increases In Glucose Uptake, etc.

This article is great [Rosenfeldt et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9794090)], and it's about uridine and not orotic acid. Orotic acid ("orotate") is a precursor of uridine but is generally toxic to the liver, in my opinion (http://scholar.google.com/scholar?q=%22fatty+liver%22+orotate+OR+orotic&hl=en&lr=), and those effects are, paradoxically, the opposite of those of uridine. Uridine has been used to treat fatty liver disease and decreases orotate formation by causing the uridine-nucleotide-mediated inhibition carbamoyl phosphate synthetase II, etc. Rosenfeldt et al. (1998) found that exogenous uridine maintained the glycogen content in the heart, increased the lactate output from the heart, and prevented much of the loss of adenine nucleotides from the heart during hypoxia. There's a typo that shows up in a couple of places, but the authors knew what they were talking about. The article is fantastic. But the concentration of uridine is listed as having been 17 mM, and the authors mean 17 uM (micromolar). The authors refer to the 17 uM concentration in the discussion section, but the mM concentration showed up in the results section. That's the Greek letter "mu," which can be an "m" in fonts other than symbol font, etc.

But the authors' analysis of the metabolic effects of uridine is really terrific. They measured the lactate output and the amount of glycogen formed and estimated that uridine had increased the rate of glucose uptake by about 50 percent but had not increased the minimal level of oxygen uptake that had occurred during the experiment. They discuss similar research and discuss the fact that the uridine-induced stimulation of glycolysis and glucose uptake (the glycogen concentration was almost double the concentration in the hearts subjected to hypoxia in the absence of uridine) is likely to have produced the uridine-induced increase in purine salvage during hypoxia. The total amount of purine nucleotides that was lost during hypoxia was almost cut in half by uridine. 17 uM is not much higher than the normal physiological plasma concentrations of uridine in humans. The major circulating pyrimidine in humans is uridine, but the major circulating pyrimidine in rats is cytidine. That's the reason the baseline plasma uridine concentration is so low in the rats. There's a lot of other research showing that nucleotides can increase glucose uptake and increase lactate output, but the effects are more complex than that.

These effects of uridine on glycogen formation and glucose uptake and even lactate output help to explain its supposed antidepressant and neuroprotective effects in humans and animals. Researchers have used uridine or its prodrugs to treat depression in a number of trials, and it's been used to treat mitochondrial disorders (encephalopathy and cardiomyopathy due to mitochondrial dysfunction, etc.). But the effects of uridine and other nucleotides are generally quite different from something like AICAR, even though other nucleotides, such as adenosine, have sometimes been shown to enhance AMPK activation (phosphorylation) and activity (by their effects of maintaining the total adenine nucleotide pool or increasing the intracellular AMP concentration more than the intracellular concentrations of other adenine nucleotides), much as AICAR activates AMPK [AMPK activation occurs when specific residues on it are phosphorylated, and the activity of AMPK is its phosphorylation of its target proteins, such as phosphofructokinase, etc.] ([Jaswal et al., 2007: (http://0-ajpheart.physiology.org.library.pcc.edu/cgi/reprint/292/4/H1978)(http://www.ncbi.nlm.nih.gov/pubmed/17172269)]. The concentration of adenosine used in that experiment was 500 uM, however, and that's a supraphysiological concentration. It's by no means a toxic concentration, because adenosine exerts various trophic effects on endothelial cells up to 1000 uM. But the extracellular adenosine concentrations don't usually exceed about 100 uM. The effects of nucleotides on AMPK activation and and activity are likely to depend on the concentrations used, and it's also important to consider the effects of nucleotides on the phosphocreatine to creatine (PCr/Cr) ratio. Many other articles show that uridine, alone or in combination with exogenous purine nucleotides, increases the PCr/Cr ratio. That effect would tend to produce allosteric inhibition of AMPK activity, etc. The main issue I have with AMPK activators is not that AMPK activation per se is "bad." In fact, the inhibition of AMPK activity or activation by specific, drug inhibitors produces toxic effects during ischemia. It's fairly clear that AMPK activation plays a role in maintaining glycolytic activity during hypoxia or ischemia. But my problem is with this assumption that "more" AMPK activation and activity is always going to be "better," and it's evident, in my opinion, that this is not always (or even usually) going to be the case, especially in the long term. This is a great article that discusses some of these issues with research on AMPK in the context of ischemia and elevated contractile activity in the heart [Dyck and Lopaschuk, 2006: (http://jp.physoc.org/content/574/1/95.full.pdf+html)(http://www.ncbi.nlm.nih.gov/pubmed/16690706?dopt=Abstract)].

Problems With Glutamine Research

A lot of these cell culture experiments showing the effects of exogenous glutamine, in the presence or absence of other substrates, are using these "luxuriant," as some authors describe "abundance" as being, concentrations of extracellular glutamine, such as 5 mM (http://scholar.google.com/scholar?q=glutamine+%225+mM%22&hl=en&lr=) or, more commonly, 2 mM (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=glutamine+%222+mM%22). Those are very high concentrations, and the extracellular fluid glutamine concentration in the brain is 400-1300 uM or so, which is 0.4-1.3 mM. I think there can be a tendency to do cell culture research and assume that cells in vivo are getting these abundant supplies of substrates, but it's not necessarily the case. I've seen that in research on magnesium, in which researchers explicitly assume that all of the ATP in vivo is going to be MgATP(2-). It's not the case, in my opinion. Also, most of the research on glutamine in humans has been done in people who have not been receiving any source of exogenous glutamine, and this is a major issue. Part of this has to do with the assumption that glutamine does not enter the brain, but there's considerable evidence that it does. The rate of efflux from the brain is almost always higher than the rate of uptake into the brain, but that says nothing about the extent to which glutamine can enter the brain. Neither does the absence of a discernable "spike" or increase in the ECF glutamine concentration, in response to the infusion of intravenous glutamine, provide any information about the extent to which glutamine has entered the brain. The glutamine-glutamate cycle is very dynamic and flexible and tends to adapt to sources of exogenous glutamine. This means that, for example, the glutamine is converted into glutamate and then either directly into 2-oxoglutarate, by glutamate dehydrogenase, or transaminated, with oxaloacetate, into 2-oxoglutarate (2-OG) and aspartate. The 2-OG can then be oxidized in the tricarboxylic acid (TCA) cycle, and its carbons can appear in all TCA cycle intermediates and in acetyl-CoA also, etc. Yudkoff et al. (1988) [Yudkoff et al., 1988: (http://www.ncbi.nlm.nih.gov/pubmed/2900878)] found that a physiological concentraion of extracellular glutamine (500 uM) caused cultured astrocytes to demonstrate no net utilization or synthesis of glutamine, and a supraphysiological concentration of 5 mM (5000 uM) was required to show a net utilization of glutamine by astrocytes. They had to remove all glutamine from the culture medium to cause the astrocytes to show a net synthesis of glutamine. The rate of synthesis is not the same thing as the rate of export, but I'm not going to get into all of that. This seems strange, but they're talking about utilization of labeled glutamine. Similarly, exogenous glutamine can spare the utilization of the existing glutamate pool for glutamine synthesis and not even elevate the total intracellular glutamate concentration [Qu et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11746415)]. These and other articles tend to suggest that, following ischemia, for example, the drastic increases in the oxidation of 2-OG are likely to cause exogenous glutamine to appear to exert no effect on the brain. I've discussed other mechanisms that suggest this, in recent postings. In other articles, researchers have noted that very few sites in the body demonstrate a *net* formation of glutamine, meaning that the rate of export from the tissue or cell group at large is higher than the rate of utilization of glutamine. A lot of research portrays the glutamine-glutamate-GABA cycle as if it's constantly generating all this glutamine and that there's endless glutamine being supplied and that cells can't oxidize more than 5 percent and can't even use all of it, because there's so much of it, or whatever. But large amounts of ATP are constantly being used up to maintain the cycle, and the amount of superfluous glutamine is likely to be quite small in many tissues, especially in trauma patients, etc.

Some Structure-Activity Relationships Among Drugs That Produce Uncoupling in Mitochondria: Are the pKa's and Log P Values Really the Key Requirements?

This is an interesting article that goes into some of the structural features [Terada, 1990: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1567840&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/2176586)] of drugs that uncouple the oxidation-reduction reactions of the respiratory chain enzymes (which are the enzyme complexes I, II, III, and IV) from the phosphorylation of ADP by F0F1-ATPase ("complex V") (this is what is meant by "uncoupling," although many authors use the term loosely and refer to other processes as being forms of "uncoupling," etc.). In my opinion, it's not just the presence of a strong electron-withdrawing group that makes these drugs' structures distinctive. I bet those drugs undergo redox cycling and generate reactive oxygen species. How could they not, in the inner mitochondrial membrane? A lot of those look like really reactive molecules. I think the equation they give, which estimates the potential for a drug to induce uncoupling, is not going to be uniformly valid, because some drugs are weak acids or weak bases and can't form these kinds of reactive metabolites that the authors discuss. Some of those drugs are like benzoquinones, basically. I wonder if excessive amounts of coenzyme Q10 can cause proton cycling across the inner mitochondrial membrane, apart from the effect of CoQ10 as a cofactor for the uncoupling proteins (and for respiratory chain enzymes, of course). Because "mild uncoupling," as discussed in many articles, reduces superoxide production in mitochondria and can be beneficial in other ways. The effects of these drugs in these articles are really at one extreme. They're extremely potent uncouplers. The requirement that the drug be relatively planar (to favor overlap of the pi orbitals, presumably, in part) to be an extreme uncoupler is probably more important than the pKa value per se, in my opinion. Here's another article that discusses some of the other types of drugs that can produce mitochondrial toxicity [Wallace and Starkov, 2000: (http://oxphos.com/staticfile/pubs/Mitochondrial%20targets%20of%20drug%20toxicity.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/10836141)]. In my opinion, a lot of the research on CoQ10 gives the false impression that it's beneficial in a wide variety of contexts in which energy metabolism has become dysfunctional. CoQ10 is vastly overrated, in my opinion, as a tool in treating mitochondrial dysfunction in people without mitochondrial disorders. Its reactivity compromises its usefulness, in my opinion, and there are plenty of situations in which increasing the activities of respiratory chain enzymes (such as through the administration of exogenous CoQ10) is, in my opinion, very undesirable and counterproductive. A lot of authors refer to all of these various cofactors or substrates (compounds that have been researched as potential treatments for disturbances in energy metabolism) as if they are equivalent or produce the same effects, and different approaches can have drastically different effects under different circumstances. This example is not even especially relevant to the problems that can occur, in my opinion, with the use of CoQ10, but, when a person or animal is healthy and experiences some injury that causes ischemia, there may be mild or no structural damage to the mitochondria at first. Days later, after the calcium influx and swelling of the mitochondria has caused structural damage, the effects of something like CoQ10, especially, could, in my opinion, become undesirable. To be useful as a neuroprotective, something should produce durable effects under a variety of conditions. If a compound's very structure can cause it to generate reactive oxygen species in a wild and unregulated way, under slightly-less-than-ideal and less-than-tidy, laboratory conditions, then the compound is not going to be especially useful, in my opinion.

Mechanisms Underlying the Mild Anticonvulsant Effects of Increases in Ketone Oxidation: Glutamate Availability, GABA Biosynthesis, and Anaplerosis

This article [Yudkoff et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11746421)] is interesting, and the authors hypothesize that increases in ketone availability to neurons, in the brain, produce their mild anticonvulsant effects by increasing the pool of glutamate that is available for GABA synthesis (this would be in GABAergic neurons, presumably, although the authors do not rule out the possibility that an increase in glutamate availability in astrocytes could increase the output of glutamine from astrocytes and thereby enhance GABA formation in neurons, etc.). Even though increases in the availabilities of beta-hydroxybutyrate and acetoacetate to the brain have been associated with antidepressant or anxiolytic and anticonvulsant effects (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=ketogenic+antidepressant+OR+anxiolytic+OR+mood), I don't think the 80% fat diet ("ketogenic diet") is a very realistic approach. The use of medium-chain triglycerides (octanoate, decanoate, etc.) has also been associated with fatty liver disease, and the entry of octanoate and decanoate into the mitochondria is not entirely carnitine-independent, as the authors of some articles have argued. Glycogen-depleting resistance exercise can elevate plasma free fatty acids (FFAs) for 2-4 days and can also elevate plasma ketone levels transiently, at least [(http://hardcorephysiologyfun.blogspot.com/2009/04/protection-against-ischemic-damage-by.html); (http://hardcorephysiologyfun.blogspot.com/2009/04/expression-of-creatine-kinase-by.html); (http://hardcorephysiologyfun.blogspot.com/2009/04/low-cholesterol-levels-and-risk-of.html)]. But circulating FFAs can be used in the biosynthesis of ketones by astrocytes, and adjacent astrocytes or neurons can then oxidize those ketones, etc. The mechanism by which ketones may produce anticonvulsant effects, as described by Yudkoff et al. (2001), is very similar to (essentially identical, to the extent that an increase in ketone oxidation can increase the pool of glutamate available for GABA synthesis by glutamic acid decarboxylase) the proposed mechanism by which exogenous glutamine can increase GABA formation in vivo in rats (http://hardcorephysiologyfun.blogspot.com/2009/03/gabaergic-effect-of-l-glutamine-in-rats.html). In my opinion, low-dose glutamine, which was used as an antidepressant augmentation approach in one small and obscure trial [Cocchi, 1976, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/03/gabaergic-effect-of-l-glutamine-in-rats.html)], could substitute for or potentially work in concert with elevations in ketones as an energy substrate for astrocytes and neurons, but, past a certain dose, the glutamine-induced reductions in plasma FFAs may begin to become counterproductive with respect to astrocyte energy metabolism. Researchers have also found that glutamine can help to preserve the adenylate charge and total adenosine nucleotide content during ischemia, in many different tissues. That may be relevant to depression, and I've discussed various aspects of purine metabolism, in the brain, etc., in many past postings [(http://hardcorephysiologyfun.blogspot.com/2009/04/increase-in-nucleotide-absorption-and.html); (http://hardcorephysiologyfun.blogspot.com/2009/04/adenosine-pka-activity-creb-activation.html); (http://hardcorephysiologyfun.blogspot.com/2009/04/research-on-use-of-creatine-monohydrate.html); (http://hardcorephysiologyfun.blogspot.com/2009/03/adenosine-and-guanosine-in-animal.html); (http://hardcorephysiologyfun.blogspot.com/2009/01/details-on-nucleotides-bioavailability.html)].

Yudkoff et al. (2001) think that the oxidation of ketones increases the intramitochondrial acetyl-CoA pool and leads to a shift in the equilibrium of the reversible aspartate aminotransferase (AA) enzymatic reaction, so as to favor glutamate formation. The authors suggest that this shift results from an increase in the consumption of oxaloacetate, one of the products of the AA reaction, by citrate synthase. Acetyl-CoA and oxaloacetate are substrates of citrate synthase, which forms citrate. The authors also note that ketone oxidation is likely to decrease the free CoA pool and thereby decrease the flux through the alpha-ketoglutarate dehydrogenase (KGDH) reaction of the TCA cycle. One issue I see is the fact that citrate synthase activity tends to be inhibited by a high acetyl-CoA/CoA ratio, but this is nonetheless a really good article. Also, they're basically saying that ketone oxidation lessens the flux of substrates through the TCA cycle (http://hardcorephysiologyfun.blogspot.com/2009/01/coenzyme-sequestration.html), given that the inhibition of the KGDH step limits the anaplerotic addition and removal of TCA cycle intermediates from the mitochondria by the malate-aspartate and malate-citrate shuttles that transfer those intermediates in and out of the mitochondria, thereby sustaining the TCA cycle. So ketone oxidation expands the pool of TCA cycle intermediates but diminishes oxidative metabolism by exacerbating the inhibition of the KGDH reaction? Are they saying ketone oxidation inhibits anaplerosis, by producing more inhibition of KGDH activity, and then increases anaplerosis by expanding the pools of citrate and citrate-derived TCA cycle intermediates that will supposedly enhance anaplerosis? The activity of AA is very high, normally, and increases in the transports of intermediates by the malate-aspartate (as discussed by the authors) and malate-citrate shuttles are not necessarily consistent with the inhibition of oxidative metabolism. It's possible that ketones buffer the pool of TCA cycle intermediates by slowing down oxidative metabolism and preventing the derangements in the cytosolic NADH/NAD+ ratio that can occur during, for example, "hyperglycolysis," following traumatic brain injuries. Then, when ketone levels fall, there's a larger pool of TCA cycle intermediates and more capacity for sustaining oxidative metabolism. What they say has a lot of truth to it, I think, but I also think that the effects of increases in ketone oxidation could be ironed out a little more.

Effects of Free Fatty Acids on ACTH and CRH Release and Responsivenes, on Sympathetic Activity, and, Potentially, Cerebral Blood Flow

This article [Migrenne et al., 2006: (http://diabetes.diabetesjournals.org/cgi/content/full/55/Supplement_2/S139)(http://cat.inist.fr/?aModele=afficheN&cpsidt=18366946)] (why is the journal Diabetes not indexed in Pubmed?) is really interesting, and the authors discuss research showing that the infusion of oleate or other free fatty acids (FFAs) into the carotid artery can cause those FFAs to enter hypothalamic neurons and either augment or decrease insulin release, in either a plasma-glucose-dependent or a glucose-independent manner, by altering the sympathetic outflow from the brain to the pancreas and other sites. The beta-oxidation of FFAs, in the hypothalamus and other parts of the brain, is required for many of these effects to occur, as discussed by Migrenne et al. (2006). This is relevant to the possibility that elevations in some saturated FFAs may produce mood-elevating or mild anticonvulsant effects, even as they may contribute to insulin resistance and other undesirable conditions, etc. [(http://hardcorephysiologyfun.blogspot.com/2009/04/protection-against-ischemic-damage-by.html)(http://hardcorephysiologyfun.blogspot.com/2009/04/low-cholesterol-levels-and-risk-of.html)]. Kok et al. (2004) [Kok et al., 2004: (http://ajpendo.physiology.org/cgi/content/full/287/5/E848)(http://www.ncbi.nlm.nih.gov/pubmed/15280154?dopt=Abstract)] cite research (reference 63) showing that high-fat diets tend to increase FFA levels, and this is fairly well-known to be the case, in my opinion. Kok et al. (2004) found that the acipimox-induced decreases in FFA levels had reduced ACTH levels in obese people. Although the authors of many articles present research to show that FFAs produce sympathetic activation or increase ACTH release, Lanfranco et al. (2004) [Lanfranco et al., 2004: (http://jcem.endojournals.org/cgi/content/full/89/3/1385)(http://www.ncbi.nlm.nih.gov/pubmed/15001638?dopt=Abstract)] found that an acute increase in plasma FFA levels reduced both cortisol and ACTH, and the authors discussed evidence suggesting the FFAs had exerted their inhibitory influence on ACTH secretion by acting on the hypothalamus (i.e. acting on neurons or astrocytes). There's some evidence that FFAs can increase sympathetic activation by acting on plasma membrane ion channels, and unsaturated FFAs can inhibit or otherwise affect beta-adrenoreceptor activation. FFAs can modify ligand binding to a number of different G-protein coupled receptors. Many of the effects on the sympathetic outflow from the CNS appear to be the result of the beta-oxidation of FFAs in the hypothalamus, though, presumably in astrocytes. Tataranni et al. (1999) [Tataranni et al., 1999: (http://www.pnas.org/cgi/content/full/96/8/4569)(http://www.ncbi.nlm.nih.gov/pubmed/10200303)] found that the elevation in plasma FFA levels after a meal correlated positively with regional cerebral blood flow (rCBF) to the dorsolateral prefrontal cortex, in association with an increase in satiety after the meal. That's a significant finding, and it could partly be a result of the beta-oxidation of those FFAs in cerebral vascular endothelial cells. The dorsolateral prefrontal cortex is obviously a site whose neuronal activity is thought to be crucially important in cognitive functioning and mood regulation, etc. [(http://scholar.google.com/scholar?q=%22dorsolateral+prefrontal+cortex%22+%22cerebral+blood+flow%22+depression&hl=en&lr=); (http://scholar.google.com/scholar?hl=en&lr=&q=%22dorsolateral+prefrontal+cortex%22+%22cerebral+blood+flow%22+cognitive)]. The recovery from depression was associated with increases in rCBF to the dorsolateral prefrontal cortex, for example [Bench et al., 1995: (http://www.ncbi.nlm.nih.gov/pubmed/7675913)]. It should be noted that something like resistance exercise tends to elevate both FFAs and ACTH and cortisol levels. The elevations in cortisol levels following resistance exercise can be very significant, and they're not really "bad," in my opinion. One can view that type of elevation as being a "strong signal" to essentially override glucocorticoid resistance at the level of the CNS or even in cells outside the brain. In asthma, for example, responsiveness to beta-adrenoreceptor agonists, which can produce anti-inflammatory effects on many cell types, can be restored within 24 hours by glucocorticoid administration. In chronic stress and depression, the issue tends not to be elevations in cortisol per se but resistance to feedback inhibition of ACTH release, by the pituitary, and CRH release from the hypothalamus. CRH generally activates noradrenergic neurons in the locus ceruleus, and an acute increase in noradrenaline availability in the hypothalamus can decrease CRH release from hypothalamic neurons [Hillhouse et al., 1975: (http://www.ncbi.nlm.nih.gov/pubmed/1079076); Valentino et al., 1988: (http://www.jneurosci.org/cgi/reprint/8/3/1016.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/3258021)]. Those types of regulatory mechanisms don't work very effectively, even under the best of circumstances, and the regulation of CRH release is very complex.

Protection Against Ischemic Damage by Free Fatty Acids in Diabetic Rats: Potential Relevance to Cholesterol Formation & Energy Metabolism in the Brain

This article [King et al., 2001: (http://ajpheart.physiology.org/cgi/content/full/280/3/H1173)(http://www.ncbi.nlm.nih.gov/pubmed/11179061?dopt=Abstract)] is interesting, and the authors found that exogenous palmitate, a saturated free fatty acid, was able to protect against damage to the hearts of diabetic rats during partial ischemia. King et al. (2001) also found that exogenous ketones were not protective. This type of research is consistent with one interpretation, as discussed previously (http://hardcorephysiologyfun.blogspot.com/2009/04/low-cholesterol-levels-and-risk-of.html), of research showing that very low cholesterol levels have been associated with higher incidences of intracranial hemorrhages/hemorrhagic strokes and deaths by suicide. King et al. (2001) found that exogenous palmitate had exerted an inhibitory influence on the overall rate of glycolysis, during ischemia, but had nonetheless been oxidized, even during ischemia, so as to produce a net augmentation of ATP production. The authors also noted that palmitate may have produced even more inhibition of the pyruvate dehydrogenase (PDH) multienzyme complex than had already been produced, in the diabetic rat hearts, from the effects of diabetes per se. The authors, additionally, discussed the fact that ketone-metabolizing enzymes are likely to be inhibited during ischemia, particularly in cardiac myocytes of diabetic rats, for various reasons. The ischemia-associated (and streptozotocin-induced) inhibition of ketone utilization may have contributed to the failure of exogenous beta-hydroxybutyrate to provide protection against ischemic/postischemic injuries (King et al., 2001).

Another specific factor the authors were getting at is that an elevation of lactate, which the authors found had occurred during ischemia, especially in the diabetic rats' hearts, tends to elevate the cytosolic NADH/NAD+ ratio, and this can, in cells with diminished oxidative capacity, lead to a decrease in the intramitochondrial NAD+/NADH ratio and inhibit enzymes whose activities are sensitive to changes in the NAD+/NADH ratio. This "translation" of the cytosolic redox state into the mitochondria occurs via the transport of substrates, such as aspartate and malate, into and out of the mitochondria, such as through the transporters that comprise the malate-aspartate shuttle. King et al. (2001) discuss research showing that the oxidation of lactose in cells from diabetic animals and humans is inhibited in a "specific" manner.

One thing that the authors may be getting at is that elevations in lactate and the lactate/pyruvate ratio, such as occur during the activation of anaerobic glycolysis during ischemia, can lead to the inhibition of the TCA cycle. An elevation in the lactate/pyruvate ratio is accompanied by an elevation in the NADH/NAD+ ratio and can, via the exchange of substrates across the outer mitochondrial membrane, decrease the intramitochondrial NAD+/NADH ratio and thereby inhibit the activities of TCA cycle enzymes. By inhibiting glycolysis, palmitate and, as the authors mention, hexanoate, may be essentially buffering the cytosolic NADH/NAD+ ratio. The authors also noted that the overall activity of the pyruvate dehydrogenase (PDHC) complex tends to be lower in the cardiac cells (and other cell types) of rats and humans with diabetes and that, in the face of a limited capacity of the cells to oxidize glucose, the exogenous palmitate had at least served as a substrate that the cells could oxidize. In other words, the PDHC activity would ideally have been higher and have allowed the cells to use glucose instead of palmitate, but the cells could only oxidize so much glucose and metabolize so much glucose, by glycolysis, without deranging the cytosolic redox state. In a context, such as the ischemic, diabetic heart, in which glucose metabolism is necessarily going to be deranged, fatty acid oxidation can evidently ameliorate the postischemic damage. King et al. (2001) also noted that other researchers had previously found exacerbations in postischemic injuries in response to exogenous palmitate, in part, as suggested by the authors, because other researchers had typically used total ischemia in their animal models. Total ischemia obviously prevents any oxidative metabolism from proceeding and prevents the utilization of palmitate or other saturated FFAs. Lloyd et al. (2004) found that the oxidative metabolism of many substrates, including palmitate, can make an important contribution to ATP production during ischemia that is even relatively severe [Lloyd et al., 2004: (http://ajpheart.physiology.org/cgi/content/full/287/1/H351)(http://www.ncbi.nlm.nih.gov/pubmed/15001444?dopt=Abstract)].

These articles [Bastiaanse et al., 1997: (http://cardiovascres.oxfordjournals.org/cgi/content/full/33/2/272)(http://www.ncbi.nlm.nih.gov/pubmed/9074689?dopt=Abstract); Bastiaanse et al., 1994: (http://www.ncbi.nlm.nih.gov/pubmed/8072018); Vauthey et al., 2000: (http://www.ncbi.nlm.nih.gov/pubmed/10822434); Olsen et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17761907); Zuliani et al., 2004: (http://www.ncbi.nlm.nih.gov/pubmed/15031316)] show that higher serum cholesterol levels or cell-membrane cholesterol levels (sarcolemmal cholesterol is plasma membrane cholesterol in cardiac myocytes) are associated with improvements in the tolerance of the cultured cells or various cell types, subjected to ischemia in vivo, to anoxia or ischemia. Three of those articles provide evidence that higher serum cholesterol levels are associated with the occurrence of less severe strokes and with decreases in the risk of post-stroke death. It's important to note that those studies do not evidently show that higher cholesterol levels reduce the risk of stroke, and it may be the case, in my opinion, that a person with "higher" cholesterol levels could have "more strokes" but have "less severe strokes," etc. The articles don't really show, in my opinion, that cholesterol is "good," and Bastiaanse et al. (1997) discuss research showing that higher plasma membrane cholesterol concentrations, in smooth muscle cells, can increase calcium influx. That would be undesirable both under "baseline," day-to-day conditions and during a stroke. Bastiaanse et al. (1997) also discuss research showing that plasma membrane cholesterol is degraded en masse, during ischemia, and that some of that cholesterol that isn't degraded is transported to the mitochondrial membranes, etc. It sounds as if some of it is, in fact, degraded to propionate and oxidized, in my opinion.

Cholesterol degradation can contribute to the propionate pool, and propionate can serve, via its metabolism into succinyl-CoA, as an anaplerotic substrate, but I'm not sure if the amounts of cholesterol-derived propionate would be large enough to contribute meaningfully to ATP production. The oxidation of cholesterol-derived propionate, a saturated, odd-chain "free fatty acid" (the term does not really apply to fatty acids that are formed intracellularly and oxidized in an autocrine manner), could conceivably help to maintain ATP production during partial ischemia. Again, I'm not sure if a significant amount would be formed from cholesterol. My guess is that it would, but I can't provide quantitative support of that supposition.

Another possibility is that the membrane cholesterol content exerts some regulatory effect on AMPK expression or on energy metabolism, etc. A lot of the research on that type of regulation has been centered around the feedback suppression of HMG-CoA reductase activity by cholesterol itself, in the liver. One reason I suggested that increases, past some critical level, in saturated FFAs as a factor that could mediate protection against hemorrhage or "suicidal-depression-associated" impairments in astrocyte energy metabolism is that most of the cholesterol in the brain is thought to be made in situ, or locally in the brain. It's conceivable that there's some combination of an impairment in the oxidation of fatty acids, derived from the blood, and intracellular fatty acid synthesis, from glucose or glutamine or other substrates, in astrocytes or cerebral capillary endothelial cells, in people who become suicidally depressed. These are just my opinions and thoughts, but it's interesting that, for example, glutamine can serve as an energy substrate for astrocytes, a role that tends to be accompanied by the transient or partial inhibition of lipolysis and beta-oxidation of FFAs, a "lipogenic" substrate, and a factor that has been shown to increase the oxidation of fatty acids [Iwashita et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16517950); (http://hardcorephysiologyfun.blogspot.com/2009/02/glutamine-decreases-plasma-free-fatty.html)]. A pronounced decrease in plasma FFAs might produce more of a decrease in brain cholesterol levels than a reduction in plasma cholesterol per se would, in my opinion. My current sense of the relationships is that a higher saturated fat intake will not especially reliably increase total serum cholesterol levels (via an increase in LDL cholesterol levels) in people with extremely low cholesterol levels, even though high saturated fat intakes do seem to be associated with higher total cholesterol levels. I would think that glucocorticoid resistance, in the context of chronic stress and depression, would impair the regulation of energy metabolism, via AMPK phosphorylation or dephosphorylation, for example, and confound a lot of these attempts to apply experimental results from nondepressed people to an understanding of the physiology at work in suicidal depression. But the presence of high cholesterol levels, from any cause, seems to be associated with higher FFA levels. Plasma ketone levels do not seem to reliably correlate with cholesterol levels, as shown in this article [Fukuda et al., 1991: (http://www.ncbi.nlm.nih.gov/pubmed/1897904)]. That's not a good example, but my point is that some kind of predictable relationship between plasma FFA levels and total cholesterol may be absent among severely depressed people, across a lower range of cholesterol levels, but may be present among people who are at the upper ranges of total cholesterol levels. And the extents to which FFAs are available for utilization (referring to the rate of uptake into the brain) or are utilized as energy substrates (referring to the rate at which they are oxidized) or cholesterol precursors by, for example, astrocytes, may not show reliable relationships with changes in the plasma FFA level.

Given the associations of depression with cardiovascular disease, it's conceivable to me that impairments in energy metabolism in the brain or cerebral vascular endothelial cells could impair the utilization of blood-borne FFAs, and so the issue might be as much about the rate of utilization of FFAs by astrocytes as it might be about the rate of uptake of FFAs into the brain. Even in the face of this poor utilization, it's conceivable that ATP production in astrocytes or endothelial cells could be very sensitive to small changes in plasma FFAs. I can't think or read about this topic any more right now. Obviously, these are only my opinions, and I'm not attempting to view any of this research in the context of any particular value system. Maybe the endothelial cells metabolize FFAs poorly in people who experience depression and allow saturated acyl-CoAs to accumulate, thereby interfering with oxidative metabolism and contributing to atherosclerosis. This accumulation could simultaneously restrict the transport of FFAs and other substrates into the brain, thereby leading to reductions in ATP production or membrane cholesterol biosynthesis in situ, etc. I think it's also likely to be important to differentiate, in research on these topics and associations, between people who have severe depression and may be suicidally depressed and those who have less severe depression. In any case, it's not a pleasant topic to think about or discuss.

Low Cholesterol Levels and Risk of Suicide or Cerebral Hemorrhage: Potential Relevance of Saturated Free Fatty Acids as Energy Substrates

There's a considerable amount of research that shows an association of very low plasma total cholesterol levels with an increase in the risk of death by suicide (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=cholesterol+suicide) or, disturbingly, by murder ("violent death") (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=cholesterol+%22violent+death%22). This is a disturbing topic, and there's also quite a bit of research that associates very low plasma cholesterol levels with an increase in the risk of hemorrhagic stroke (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=cholesterol+hemorrhage). The arguments that researchers have made, in their attempts to explain these associations, have not been very compelling to me, for the most part. Many people seem to have bought in to the idea that low omega-3 fatty acid intake was some sort of "surrogate marker" for low plasma cholesterol and that the apparent increases in suicidality, in the context of low cholesterol levels, are actually a result of low omega-3 intakes. This makes no sense, in my opinion, and it's never made any sense to me. That explanation for the association of low cholesterol with suicide would, in fact, directly conflict with other research that relates to the association of low cholesterol with hemorrhage. Specifically, researchers have found a positive association of high omega-3 fatty acid intakes with risk of hemorrhage (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22omega-3%22+hemorrhage), and researchers have also found that low saturated fat intakes are associated with an increased risk of hemorrhage (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22saturated+fat%22+hemorrhage). (Note that researchers sometimes use the term "intraparenchymal hemorrhage" to refer to an intracranial hemorrhage into the central nervous system parenchyma, as opposed to the hepatic, or liver, parenchyma, etc.)

I think all of these associations could be explained in terms of decreases in saturated free fatty acid availability to the brain and endothelial cells lining the blood vessels supplying the brain. Saturated free fatty acids behave quite differently from the ways unsaturated fatty acids behave, and there's considerable research showing that elevations in free fatty acids, following exercise, contribute strongly to glycogen replenishment in the liver and skeletal muscles. High-intensity exercise can elevate fasting plasma free fatty acids for four or more days post-exercise. Kiens et al. (1998) [Kiens et al., 1998: (http://ajpendo.physiology.org/cgi/content/full/275/2/E332)(http://www.ncbi.nlm.nih.gov/pubmed/9688636)] found, similarly, that fasting FFAs (note that the FFA measurements in the evening, at 30 hours post-exercise, was approximately a fasting measurement but might have been expected to be even higher in the true, fasted state, meaning in the morning) were still elevated at 42 hours post-exercise. And strength training, performed correctly, as a form of high-intensity exercise, generally produces the greatest elevations in FFAs. There's increasing evidence that impairments in cellular energy metabolism may be at the root of some psychiatric conditions, such as depression, and astrocytes and endothelial cells are capable of oxidizing palmitate and other free fatty acids, in addition to ketones, as energy substrates. Additionally, there is research showing that fasting is beneficial to animals with traumatic brain injuries (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22Fasting+Is+Neuroprotective+Following%22). Everyone assumes that this is because fasting can elevate ketones (acetoacetate and beta-hydroxybutyrate, primarily), and ketones have been shownt to exert neuroprotective effects in many articles (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=ischemia+%22beta-hydroxybutyrate%22+OR+acetoacetate). But my sense is that ketone levels do not reliably correlate with plasma cholesterol levels. I'm not sure if *saturated* free fatty acid levels correlate with plasma cholesterol levels, but my sense is that the proportion of saturated free fatty acids would increase as plasma cholesterol increased. Saturated fatty acids are preferentially used for cholesterol biosynthesis, in comparison to monounsaturated (and, obviously, omega-3) fatty acids. Also, fasting for even short periods of time elevates plasma free fatty acids, but I think ketones do not necessarily become elevated to very significant levels until one has fasted for a fairly prolonged period of time. My reason for not focusing on ketones is that I've seen articles showing very inconsistent relationships between serum ketone levels and serum cholesterol levels, but this does not mean that ketones would not be important as energy substrates for neurons and astrocytes. But even the overnight fast, during sleep, elevates free fatty acids substantially, an effect that is thought to partly be due to the nighttime growth hormone release. Nonetheless, high-intensity exercise elevates both FFAs and ketones [Walsh et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9562294)]. But the assumption in the research looking at associations of factors with plasma cholesterol did not, in general, assume that the people were exercising, etc. And increasing one's omega-3 fatty acid intake is thought to potentially increase ketone formation (by the liver, etc.), and that's not an effect one would expect to see if, as I'm assuming in the context of this discussion, an increase in omega-3 intake and a decrease in saturated fat intake were predisposing to hemorrhage [Freemantle et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16829066)]. The authors of many of the articles on fat intake have suggested that the supposed protective effect of saturated fat intake, in the context of the risk of hemorrhage, might have more to do with the ratio of saturated fat to omega-3 fats. In other words, a person who eats more saturated fats will, at a given caloric intake and dietary composition, tend to eat fewer grams of omega-3 fats and have a higher percentage of saturated fatty acids in erythrocyte membrane phospholipids than a person who eats fish three times a day would, etc. Other researchers have suggested that a higher saturated fat intake simply increases cholesterol levels and thereby produces the supposed protection against intracranial hemorrhage. Although saturated fats (palmitate, etc.) are generally thought to be utilized more for cholesterol biosynthesis than many other fatty acids, I don't think that small increments in saturated fatty acid intake (I think even something like an increment, or increase, of 18 grams per day of saturated fats was found to be associated with protection from hemorrhage in some articles, and that's not some kind of enormous intake level) would produce large and incremental increases in plasma cholesterol. That's my sense of it, at least. But increasing the ratio of dietary saturated fat to polyunsaturated fats would, in my opinion, be expected to increase the "percentages" of erythrocyte phospholipids containing saturated fatty "acyl" side chains and, by extension, in my opinion, increase the saturated fraction of the plasma FFA pool.

Additionally, one explanation for the association of low cholesterol with hemorrhage was that a low cellular cholesterol content in smooth muscle cells made those cells less resistant to anoxia or hypoxia. Why would this be? Perhaps, during hypoxia or ischemia, cholesterol is degraded more extensively to propionate (cholesterol is, in fact, a source of odd-chain fatty acids, such as propionate, that can be metabolized to succinyl-CoA and thereby serve as anaplerotic substrates). I can't find the article discussing that, but I remember it. The researchers were saying that cerebral ischemia can produce localized, smooth-muscle-cell necrosis ("arterionecrosis"), by diminishing blood flow to a section of an artery (although, one would think , in my opinion, that low cholesterol would be associated with venous hemorrhage, also, and not just or even mainly with arterial hemorrhages), and that an "adequate" smooth-muscle-cell cholesterol content would reduce that arterionecrosis. The localized, necrotic death of smooth muscle cells is one explanation for reperfusion-induced or reperfusion-associated hemorrhage. It's possible that there is not a great deal of validity to that "explanation" for the association of low cholesterol with hemorrhage (the researchers' concept that a low smooth muscle cell cholesterol content might decrease the resistance of those cells to hypoxia/anoxia), and my suggestion that a "cellular-cholesterol-content-induced" increase in propionate oxidation may also not be valid. But finding evidence that would contradict those concepts would not negate the possibility that increases in, for example, the "area under the curve" for saturated, plasma free fatty acids could, for example, be associated with less depression or with improvements in the maintenance of astrocyte glycogen contents at different times during the day or during recovery from exercise, etc. There can be a tendency to view all free fatty acids as being always bad, and one could argue that this tendency has more to do with value-laden dogma about physiological processes being "good" or "bad" ("good fats" vs. "bad fats," etc.). If all free fatty acids, including palmitate and other saturated free fatty acids, are bad for cellular energy metabolism all the time, then why can resistance exercise, which elevates plasma free fatty acids significantly for prolonged periods of time but simultaneously tends to improve insulin sensitivity (elevations in FFAs normally are associated with a worsening of insulin sensitivity) and increase the capacity of skeletal muscle cells and other cell types to oxidize those fatty acids, produce improvements in mood in some people [Doyne et al., 1987: (http://www.runningtherapie.nl/Portals/0/ccp555748.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/3454786)]? I think an important distinction needs to be made, from the standpoint of energy metabolism, between saturated free fatty acids and unsaturated free fatty acids (a.k.a. nonesterified fatty acids). Saturated fatty acids do not produce all of the same inhibitory effects, on the activities of enzymes and on the binding of ligands to receptors, as unsaturated fatty acids produce. In many cases, saturated fatty acids do not produce any of the less-than-desirable "regulatory" or toxic effects, at least in experiments performed in vitro, that unsaturated fatty acids produce.

I'm not saying that elevating saturated free fatty acids is likely to be a particularly good strategy in any particular disease context, but, for example, endothelial cells in some blood vessels are known to depend on either glutamine or free fatty acids, to a significant extent, as energy substrates, particularly in the fasted state. Also, I think that inappropriate elevations in saturated plasma free fatty acids could reasonably be expected to contribute to atherosclerotic disease, particularly if those elevations occurred outside of the context of something like resistance training or some other form of exercise. But if the role of saturated free fatty acids could be investigated in some of these contexts that I've discussed, researchers might be able to develop alternative substrates for maintaining cellular energy metabolism, such as in astrocytes and neurons, and avoid the problems associated with beta-oxidation of fatty acids in the cells of adults. Beta-oxidation is not a particularly efficient or "clean" process and tends to be problematic, from the standpoint of the flux of substrates through the tricarboxylic acid cycle, etc. (problematic from the standpoint of carbohydrate metabolism). Nonetheless, I would argue that there is a need to face some of these disturbing associations. To acknowledge that free fatty acids may influence astrocyte energy metabolism in some positive ways, as, in my opinion, they may, is not to say or imply that this is "good" or that people should eat massive amounts of saturated fats or try to elevate free fatty acids artificially, outside of the context of something like strength training/resistance exercise. Rather, the idea, in my opinion, could be to research alternative cellular energy substrates, such as glutamine, etc., in those contexts.

Protection Against Postischemic Damage by Uric Acid and Evidence Arguing Against a Strongly Destructive Role for Xanthine Oxidase Activity

This article [Mink and Johnston, 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17335786)] shows that the intravenous infusion of 600 umol/kg/hr of hypoxanthine (HPX) reduced damage to the brain following experimental hypoxia and cerebral ischemia (8 minutes of each). The authors compared the degree of damage in the group that had received HPX, given during the interval of 30-minutes before and 30 minutes after the induction of ischemia (the 30 minutes "after" had included the 16 minutes during which the animals had been subjected to hypoxia and then ischemia), to the degrees of damage in the groups that had received either an infusion of xanthine (XAN) or an inactive vehicle. The relative degrees of damage were not significantly different in the groups that had received either xanthine or the vehicle, and the authors noted that these data, along with other data, argue against the notion that free-radical production from xanthine oxidase activity contributes strongly to damage following cerebral ischemia. The authors also found that the cerebral HPX levels, measured after the four hours of reperfusion had elapsed, were actually much lower in the group that had received the HPX infusion than in the other groups. Given that these decreases in the post-reperfusion HPX levels had been accompanied by reductions in postischemic brain damage, the authors noted that the HPX had probably been salvaged heavily or converted into xanthine, etc. Some of the XAN in the XAN group may have also been used for salvage (XAN can be salvaged to xanthosine and guanosine).

These results are important and suggest, as noted by the authors, that the protective effects of allopurinol, a xanthine oxidase inhibitor used to treat hyperuricemia, against cerebral ischemia are mediated by mechanisms other than XO inhibition per se. In other words, the reduction in uric acid is probably not protective in and of itself (especially since uric acid has been shown in many studies to protect against experimental, ischemic brain injuries) and, more importantly, the xanthine oxidase activity per se seems unlikely to be a major source of oxidative damage to the brain. The authors provided more XO substrates, which are HPX and XAN, and did not find increases in ischemic damage. The authors noted that the high Km value for XAN binding to XO (88 uM) as a substrate means that XO was not saturated with XAN during the experiments and wasn't in untreated animals either (the brain XAN content in the cerebral cortex of the rabbits was only 18 uM at 30 minutes after the initiation of reperfusion). This means that exogenous XAN is likely to have been utilized as a substrate for XO, as noted by the authors, but also means that the XO activity in the control group might not have necessarily been lower (the XO enzymes in the control rabbits would presumably have had access to plenty of endogenously-produced XAN, etc.). There's an article showing that allopurinol can exert antinociceptive effects that are evidently due to elevations in the cerebrospinal fluid guanosine and adenosine levels, and some of the antinociceptive effects can be blocked by adenosine receptor antagonists [Schmidt et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19133997)]. Those types of elevations in CSF guanosine and adenosine could also explain the neuroprotective effects of allopurinol. Even though the paper by Mick and Johnston (2007) doesn't necessarily preclude a damaging effect of XO-derived reactive oxygen species, the evidence, in my opinion, that XO activity is "all-bad" or that uric acid is a mediator of ischemic damage is not compelling at all, to say the least. Betz et al. (1991) [Betz et al., 1991: (http://www.ncbi.nlm.nih.gov/pubmed/1996699)] also noted that XO activity is unlikely to make a large contribution to oxidative damage following ischemia. To the contrary, uric acid has been shown to protect against postischemic damage, in many articles, in both humans and animals [here are a couple of the articles that show or discuss this: Yu et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9726432); Amaro et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18271711); Chamorro et al., 2004: (http://www.ncbi.nlm.nih.gov/pubmed/14962621); Amaro et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17525395)(http://stroke.ahajournals.org/cgi/reprint/38/7/2173.pdfhttp://stroke.ahajournals.org/cgi/reprint/38/7/2173.pdf); Romanos et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/16596120); Teng et al., 2002: (http://www.ncbi.nlm.nih.gov/pubmed/12398932); Keller et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9425011)]. Uric acid and xanthine have both been shown to exert fairly strong feedback inhibition of XO activity [Rubbo et al., 1991: (http://www.ncbi.nlm.nih.gov/pubmed/1653611); Radi et al., 1992: (http://www.ncbi.nlm.nih.gov/pubmed/1322703)], and this feedback inhibition appears to be significant at normal, physiological concentrations in humans [Tan et al., 1993: (http://www.ncbi.nlm.nih.gov/pubmed/8134172)]. But the feedback inhibition has actually been shown to increase superoxide production by XO in vitro (by the isolated enzyme) (Rubbo et al., 1991; Radi et al., 1992). At the same time, Tan et al. (1993) found that 150 or 300 uM uric acid reduced the production of superoxide in human plasma overall by 23.2 and 32.0 percent, respectively. This indicates, in my opinion, that the overall effect of uric acid, despite its potential to increase superoxide production by XO, is to decrease superoxide formation. So elevations in uric acid levels, produced by exogenous purines or exogenous uric acid, during ischemia protect against ischemic damage, may increase free radical production by XO, but appear to decrease superoxide formation in the blood overall. This is consistent with the results of many other articles. The articles I've discussed provide more evidence, in my opinion, that uric acid per se and XO activity per se should not be viewed as being major factors, in the absence of exogenous uric acid or purine precursors of uric acid, contributing to oxidative damage following ischemia.

When one considers the nearly countless articles showing protection by uric acid against experimental autoimmune encephalomyelitis (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=encephalomyelitis+uric+OR+urate) and mitochondrial damage (http://scholar.google.com/scholar?q=mitochondrial+peroxynitrite+uric+OR+urate&hl=en&lr=), etc., these association studies, implying that uric acid is "independently" damaging, become bizarre to see, in my opinion. Hozawa et al. (2006) noted that elevations in serum urate are a risk factor for stroke but that uric acid itself "may" not cause strokes [Hozawa et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16239005)]. It's important to remember that ischemia itself (meaning ongoing, intermittent, low-level cerebral ischemia or ischemia in blood vessels outside the brain) increases purine nucleotide export and XO activity and uric acid production in a very reliable manner. It's worthwhile to remember that statistic-driven association studies are not a substitute for reasoning.