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.

Relationships of Intracellular Free Magnesium to the Cytosolic Phosphorylation Potential and Rate of Mitochondrial ATP Synthesis

Jacobsen et al. (2001) [Jacobsen et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11431727)] found that the intracellular free magnesium (meaning Mg2+, abbreviated Mg, that was not bound to proteins or complexed with nucleotides) concentrations, in the skeletal muscles of people who exhibited cirrhosis, correlated positively with the maximal rates of ATP formation that the authors measured, using 31P-MRS, after the people had just finished exercising. The authors estimated the intracellular free Mg levels by taking into account the intracellular pH and looking at the difference between the chemical shift of alpha-ATP, or alpha-NTP (nucleotide triphosphates, which are assumed to consist primarily of ATP), and the shift of beta-ATP/beta-NTP's. Heath and Vink (1999) [Heath and Vink, 1999: (http://jpet.aspetjournals.org/cgi/reprint/288/3/1311)(http://www.ncbi.nlm.nih.gov/pubmed/10027872)] found that intravenous Mg increased and thereby normalized the cytosolic phosphorylation potential (CPP) in rats that had been given experimental brain injuries, and the intracellular free Mg concentration and CPP values both correlated positively with markers that were indicative of favorable neurological outcomes. The CPP = [sigma sum of ATP anions]/ [sigma ADP] [sigma Pi], and the calculation of the ADP species requires one to take into account the intracellular pH and free Mg levels. The sum of the ATP species includes MgATP(2-) and ATP(4-), and [sigma ADP] includes the concentrations of MgADP(-) and ADP(3-) but also takes into account the influence of the Mg availability on the overall, intracellular creatine kinase equilibrium that is a reflection of the mitochondrial and cytosolic equilibria. I'd like to know the assumptions that the authors made about the relative abundances of MgATP(2-) and ATP(4-). It's not clear to me that the authors are using the intracellular Mg concentration as a basis for estimating the relative amounts of MgATP(2-) and MgADP(-), in relation to the free nucleotides. I've seen some authors assume that most or all of the ATP exists as MgATP(2-), and this is unlikely to be the case in the cells of most humans, in my opinion. I get the impression that Heath and Vink (1999) only took into account the shift that an increase in Mg availability produces in the overall creatine kinase equilibrium. Mg tends to shift the eqilibrium constant to increase the phosphocreatine/creatine ratio at equilibrium. But the Mg-induced increase in the CPP could have partially resulted from increases in the proportions of MgATP(2-) and MgADP(-) (meaning that more total ADP would be available and would allow for more total ATP) and not just from an effect of Mg on ADP, etc. I've seen other authors argue that the increases in the CPP that occur in association with increases in free Mg are not desirable in the context of presumably- or definitively-chronic mitochondrial dysfunction in the brain, as in people who have cluster headaches mitochondrial disorders resulting from mutations in the nuclear or mitochondrial genomes. The argument by some of those authors has been that an increase in the CPP may be associated with an increase in oxidative stress, given that a higher CPP is indicative of a high rate of ATP turnover. Heath and Vink (1999) found that, in the period shortly after a traumatic brain injury, the ATP levels were not decreased. That might be one reason for the fairly clear benefit of Mg. Although there is the potential for Mg to cause some strange effects that are not always going to be beneficial, a large amount of research has shown that Mg depletion is harmful to the brain and that Mg repletion tends to be beneficial, in my opinion. The Mg-induced, transient decreases in blood pressure or Mg-induced decreases in the peripheral vascular resistance could be less-than-beneficial after brain injuries, in some cases. Mg-induced peripheral vasodilation could reduce venous return by decreasing the sympathetic outflow from the CNS, and a decrease in venous return to the heart could tend to reduce the cardiac output and thereby reduce cerebral blood flow in some patients. In some people who have had brain injuries, the regional cerebral blood flow can be dependent upon and positively correlated, up to a point, with the cardiac output or mean arterial pressure ("pressure-passive" autoregulation of cerebral blood flow, etc.). It's possible that that type of dependence could show up, to a lesser degree, in some people who have psychiatric disorders, in my opinion, or in chronic fatigue syndrome that is accompanied by orthostatic tachycardia or hypotension (orthostatic tachycardia usually indicates that the sympathetic activity is decreased, in my view). In those cases, Mg could help up to some individualized point or dosage but could then become counterproductive as one kept increasing the dosage, because of reductions in venous return or mean arterial pressure or because of other mechanisms, such as Mg-induced, excessive increases in cytosolic 5'-nucleotidase activities, etc. But then, in the longer term, one might expect Mg repletion to reduce that kind of abberant regulation of cerebral blood flow. Pressure-passive autoregulation can result from vasospasm, in which there's localized vasoconstriction that persists in the face of the increases in shear stress that would normally produce vasodilation, etc. The smooth muscle cells of cerebral arteries are exceptionally sensitive to changes in calcium influx, and that's one reason Mg, as a mild calcium channel antagonist, is thought to produce prominent cerebral vasodilatory effects. The antithrombotic effects that Mg can exert could also gradually cause the regulation of regional cerebral blood flow to become less dependent on changes in the mean arterial pressure or cardiac output. But some of the "sympatholytic" effects of excessive amounts of Mg could become counterproductive in ways that might not be overcome by antithrombotic or cerebral vasodilatory effects of Mg. I get the feeling that a lot of people find it disturbing to think that some cases of severe depression or chronic fatigue syndrome are partially a result of reductions in regional cerebral blood flow (and that increasing cerebral blood flow might ameliorate those symptoms), but, in my opinion, it's very likely to be the case. Look at the association of migraine with depression or whatever else. The authors of one of those MRS studies of the effects of SAM-e, with its measly effects on the intracellular adenosine nucleotide pools in endothelial cells and neurons and on the perivascular interstitial fluid adenosine levels, found some evidence that SAM-e might have increased cerebral blood flow in a subset of people with depression. Obviously, ATP disodium could reasonably be expected to increase the regional cerebral blood flow in parts of the brain in which the cerebral blood flow is reduced. But that's just my opinion. But there has to be some mechanism to account for the magnitude of the effects of something like that, and, in my opinion, whatever effects may occur are either going to be a result of AMP- and ADP-stimulated respiration or glycolytic activity (and, consequently, glucose uptake) or of increases in cerebral blood flow or both or similar "secondary" effects on energy metabolism. In other words, any increases in ATP levels or in the rates of ATP turnover would probably not be only a result of increases in the overall pools of adenosine nucleotides per se, independent of the secondary changes in glucose consumption or uptake or of oxygen uptake, etc. I say that because the effects of adenosine really can't be accounted for by the capacity of its ribose moiety to serve as a precursor of glycolytic intermediates, as shown by some of the research I've cited in past postings.

Phosphate (Pi) Sequestration by Fructose; Potential Effects of Changes in Pi Availability on the Mitochondrial Proton Gradient and on XDH Activity

This is one of the other articles that includes a discussion of the mechanisms by which fructose acutely increases plasma uridine and also urinary uridine excretion [Yamamoto et al., 1997: (http://www.ncbi.nlm.nih.gov/pubmed/9160822)], but Yamamoto et al. (1997) didn't show the decreases in plasma uridine, to levels below the baseline concentrations, that occur after the increases (see a recent posting). Yamamoto et al. (1997) also didn't address the mechanism by which the fructose-induced inorganic phosphate (Pi) sequestration leads to purine degradation, but a key mechanism is that the decrease in intracellular Pi disinhibits adenosine monophosphate (AMP) deaminase. AMP deaminase is normally inhibited by Pi. Yamamoto et al. (1997) cited a lot of interesting research, however. They suggested that the ethanol-induced (and, by less direct mechanisms, fructose-induced) increases in hypoxanthine and xanthine might have resulted from the elevations in the cytosolic NADH/NAD+ ratio that results from the metabolism of ethanol to acetaldehyde, given that NADH inhibits xanthine dehydrogenase activity. Fructose could also produce that effect, albeit to a lesser extent than ethanol. In addition to the ATP depletion that ultimately can occur through the disinhibition of AMP deaminase, resulting from fructose-induced Pi sequestration, Yamamoto et al. (1997) referred to the direct consumption of ATP in the fructokinase reaction that forms fructose-1-phosphate and thereby sequesters Pi [see also Phillips and Davies, 1985: (http://jp.physoc.org/content/520/3/909.full)(http://www.ncbi.nlm.nih.gov/pubmed/2992452)]. It's worth noting that fructose also depletes guanosine triphosphate (and guanosine nucleotides in general, as shown in multiple articles), partly because fructokinase activity is apparently GTP-dependent (Phillips and Davies, 1985). Fantastic. It depletes all the major nucleotide pools. Cytidine depletion would also be expected to occur (I'll bet there's some research showing that, too), given that cytidine is formed from uridine. But the point I was going to make is that changes in intracellular Pi could regulate xanthine dehydrogenase activity by buffering the intracellular pH, given that increases in the intracellular pH tend to activate phosphofructokinase and glycolytic activity overall. That increase in glycolysis would then increase the NADH/NAD+ ratio and reduce xanthine dehydrogenase activity, and that could conceivably allow for more salvage of hypoxanthine (and even xanthine, which can be salvaged to a minimal extent by a two-enzyme pathway). Yamamoto et al. (1997) cited research showing that lactate can decrease the rate of urinary uric acid excretion but apparently doesn't reduce the excretion of hypoxanthine or xanthine [the oxypurines that Yamamoto et al. (1997) are referring to]. Does Pi repletion increase or decrease ischemia-induced glycolytic activity? Pi repletion generally does increase the activities of glycolytic enzymes, in many of the articles I've seen, but it could also reduce the kinds of wild fluctuations in the intracellular pH that can occur during ischemia. The Pi-induced increases in glycolytic activity by allosteric mechanisms could increase the cytosolic NADH/NAD+ ratio [Zhou et al., 2005: (http://jp.physoc.org/content/569/3/925.full.pdf+html)(http://www.ncbi.nlm.nih.gov/pubmed/16223766?dopt=Abstract)] and inhibit xanthine dehydrogenase activity (meaning that, from a simplistic standpoint, that effect could decrease uric acid formation and enhance purine salvage, conceivably), and, in the absence of a high intake of a phosphate salt displaying an abnormal ratio of monobasic to dibasic orthophosphate (orthophosphate refers to [HPO4(2-) + H2PO4(-) + the less-than-1-% contribution of PO4(3-)]), Pi repletion can produce an alkalinizing effect that could also activate glycolysis and further reduce xanthine dehydrogenase activity. But it could also exert more of a neutral effect. Those are just speculative thoughts.

For that matter, I wonder if the alkalotic effects of excesses of Pi might abolish or decrease the mitochondrial proton gradient under some circumstances, by mimicking the effects of uncouplers. Pi could conceivably stimulate respiration by that mechanism [that commonly occurs as a compensatory response (http://scholar.google.com/scholar?hl=en&q=stimulate+uncoupler+mitochondrial+respiration)], and that could explain those articles I cited, in a past posting, showing that Pi can increase the postprandial metabolic rate in humans, etc. That could conceivably account for some of its supposed psychiatric or psychoactive effects, and the "pseudodepression" and other effects of Pi depletion could be due to the poor "regulation" of the mitochondrial membrane potential. There are all sorts of articles showing that the stimulation of respiration is associated with phosphate influx into mitochondria, and phosphate influx interacts with ADP-stimulated respiration, etc. The point is that the effects of different concentrations of intracellular or intramitochondrial Pi on respiration could conceivably be either "bad" or "good," depending on the way you look at the effects.

It would be interesting to see some in vivo research on the effects of Pi depletion or repletion on the exercise-induced loss of purine nucleotides, for example, because it could be a complex set of effects. It's interesting that Hellsten et al. (1999) [Hellsten et al., 1999: (http://jp.physoc.org/content/520/3/909.full)(http://www.ncbi.nlm.nih.gov/pubmed/10545153?dopt=Abstract)] argued that the initial effect of exercise had been to increase Pi availability, thereby inhibiting AMP deaminase activity, but that the decreases in intracellular pH that had subsequently occurred had activated AMP deaminase activity. It's interesting that an increase in the inhibition of AMP deaminase by Pi would tend to lead to a relative increase in adenosine availability, and some of that adenosine would presumably serve to increase blood flow to the exercising muscles. I wonder if that increase could lead to a greater loss of adenosine, however, or if the Pi-mediated inhibition of AMP deaminase activity (as in the endothelial cells in which much of the adenosine deaminase-mediated deamination of interstitial-fluid adenosine occurs) would mean that more adenosine could be released and then also salvaged. The intracellular and extracellular adenosine concentrations are not usually very different, and there's a slight, inwardly-directed, transmembrane adenosine gradient. Usually, one thinks of adenosine release as being a unidirectional process that's "coupled" to an increase in the degradation, by adenosine deaminase in endothelial cells, of the adenosine to inosine and hypoxanthine. But, presumably, that's not always going to be the case. It's interesting that uncouplers are used to increase extracellular adenosine concentrations [see the reference to "respiratory uncouplers" on the first page of Rubio et al., 1972: (http://www.ncbi.nlm.nih.gov/pubmed/5022662)], and my overall point is that excessive concentrations of intracellular Pi, to the extent that they are achievable, could conceivably have some adverse effects that would go beyond the well-known increases in the risk of calcification, etc.

Interactions of Phosphate and Calcium Homeostasis with the Coagulation Cascade: Potential Relevance to Depression and Other Psychiatric Symptoms

So the "bottom-line," "take-home" message of that last posting is that, in susceptible individuals or individuals in whom the coagulation cascade has been transiently or mildly activated by infectious mono or influenza, an increase in serum calcium within the normal range could produce depression or psychiatric symptoms by producing low-level thrombogenic effects (effects that essentially disturb mitochondrial functioning, as the feed-forward activation of the coagulation cascade essentially always does), and reducing serum calcium by reducing the dietary calcium or vitamin D intake could ameliorate those effects. Increasing the ratio of the phosphate to calcium intake could be a superior way of addressing those potentially-thrombogenic effects (and calcium influx promoting effects, in neurons) of increases in serum calcium. And idiosyncratic effects of glutamine supplementation might be addressed by decreasing the vitamin D or calcium intake or increasing the relative intake of phosphate, to some small extent, given the potential for slight "calcemic" and hypophosphatemic effects of glutamine. It's possible that an increase in serum phosphate would reduce calcium influx into platelets, given that increases in phosphate availability have reduced stimulus-induced intracellular calcium influx in beta-cells, for example, if memory serves (see past postings), and in other cell types. That's thought to be one mechanism underlying magnesium's antithrombotic effects (and relative absence of hemorrhagic effects).

I don't have time to go into the research, but, in my opinion, some of the research that would seem to rule out a role for the activation of the coagulation cascade in depression (http://scholar.google.com/scholar?q=coagulation+psychiatry&hl=en) does not rule it out, given that research in people with lupus and research on the coagulation cascade in general have shown that localized endothelial cell activation, such as in cerebral blood vessels, can occur and can cause localized microthrombi or low-level thromboses without producing measurable changes in the systemic coagulation parameters. Blood tests of coagulation parameters are notoriously insensitive and problematic, in my opinion. This is not a scientific statement, but, if it were possible to easily evaluate coagulation function, then monitoring people on warfarin wouldn't be so difficult and complex for both doctors and patients (the people taking warfarin, etc.), in my opinion. The coagulation cascade is extremely complex, and quantitative data on coagulation parameters are not going to tell one all that much about the individual and tissue-restricted effects of that state in any one person. The INR, for example, is very insensitive and displays a semi-logarithmic relationship with changes in the serum prothrombin levels, etc. I tend to think the ex vivo tests on platelet function are also not always going to have relevance to tissue-restricted (or endothelial-site-restricted) thrombogenic effects in the brain, for example. Benign intracranial hypertension/idiopathic intracranial hypertension commonly produces psychiatric symptoms, but that type of disease state may just be a slightly more extreme state along a spectrum of low-level thrombogenic changes that could potentially contribute to some forms of severe depression or chronic fatigue syndrome, etc. Those articles about visual dimming in depression could also indicate that low-level activation of the coagulation cascade is occurring, given the common occurrence of visual dimming in idiopathic intracranial hypertension (and the fact that idiopathic intracranial hypertension is thought to be not-infrequently caused, in part, by venous sinus thrombosis). Magnesium can also produce antithrombotic effects and may, in my opinion, be a lot less likely to cause bleeding, upon adjustment to a dose increase in 1-2 days, than many or most of the many other compounds that influence platelet activation and the coagulation cascade. In any case, it's worthwhile to remember that the use of Ginkgo biloba extracts has been associated with intracranial hemorrhages in many case reports (http://hardcorephysiologyfun.blogspot.com/2008/12/ginkgo-biloba-extracts-and-intracranial.html), and many compounds can reduce coagulation by mechanisms that could be very dangerous and unpredictable. So one would want to talk to one's doctor about this type of thing. Purines have produced antithrombotic effects in a lot of animal studies and appear to be a lot less likely to cause bleeding than most of these other physiological approaches, but that's just my opinion, based on my experiences during infectious mono, several years ago. These are all, obviously, just my opinions.

Case Reports of Myopathy in Hypophosphatemia

It's easy to look at this article [Schott and Wills, 1975: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=491911&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/1151410)], in which Schott and Wills (1975) described a person who had had hypophosphatemia and, as a result, had become almost unable to walk from muscle weakness, and say that this type of thing couldn't occur in the present day. But this type of thing could, arguably, more easily happen in today's medical system than in 1975 (especially if someone described the symptoms in a slightly different way). It's worth noting that the authors, Schott and Wills (1975) were not the ones who had, on two occasions, dismissed the person's symptoms as having been "hysterical" in nature. Before Schott and Wills (1975) saw the person, some other doctors had seen her and had not been able to find anything objectively wrong with her. Today, a doctor might only be able to spend ten minutes with his or her patients, because of insurance issues and everything, and refer to the complaints of back pain and muscle weakness as being "somatic symptoms," etc. I don't usually do this, but here are a couple of excerpts:

"Also at that time she became aware of muscular weakness, initially causing difficulty in climbing high steps and in manoeuvring her legs when getting into a car. The weakness of both thighs subsequently progressed, and was associated with intermittent hip and low back pain. She was investigated in another hospital one year before admission here, when proximal muscle weakness and a waddling gait were noted, together with brisk tendon reflexes and bone tenderness on palpation. An electromyogram performed at that time was normal, and the only significant abnormal investigation found was a low renal threshold for glucose. The symptoms were considered to be hysterical and she was discharged. Her weakness, however, became more profound, and she had to pull herself upstairs, a task that became increasingly difficult with the development of proximal weakness in the arms, and for six months before admission she had been unable to raise her arms above her shoulders. She was admitted at that time to a second hospital for investigation, and was again thought to be psychoneurotic and no specific therapy was prescribed. She continued to deteriorate, commenced walking with a Zimmer frame, and was admitted to this hospital for further assessment. On direct questioning, she had noted that her nails had become brittle, and that she had had a tendency to vomit occasionally over the preceding 10 years. Her weight had fallen by about 13 kg over four years, although she had always eaten an adequate and normal diet. Her sister reported that the patient had 'shrunk' over the preceding two years" (Schott and Wills, 1975, p. 298).

The muscle weakness could have been partly a result of neuropathy induced by phosphate depletion [(http://scholar.google.com/scholar?hl=en&q=phosphate+hypophosphatemia+neuropathy+OR+neuropathic); (http://scholar.google.com/scholar?hl=en&q=phosphate+muscle+weakness+hypophosphatemia+neuropathy+OR+neuropathic)], given that the authors of some of those articles, in the search results, have described neurological problems resulting from phosphate depletion. The muscle weakness and exercise intolerance that can occur in phosphate depletion [(http://scholar.google.com/scholar?hl=en&q=phosphate+hypophosphatemia+exercise+intolerance); (http://scholar.google.com/scholar?hl=en&q=phosphate+muscle+weakness+hypophosphatemia)] are reminiscent of the types of symptoms that people experience in mitochondrial disorders, as discussed in past postings. Some interesting articles came up in those searches. I've only looked at the abstracts so far, but the authors of this one described a person who had had fatigue and exercise intolerance that were suggestive of some mitochondrial or bioenergetic pathology [Land et al., 1993: (http://www.ncbi.nlm.nih.gov/pubmed/8400863)]. This is another one showing the association of phosphate depletion with poor insulin sensitivity [Haap et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16391583)], and Haap et al. (2006) found that serum phosphorus correlated positively with a marker of insulin sensitivity. The authors mention, in the abstract, that one can't definitely say that the higher phosphate availability causes the cells to become more responsive to insulin, but it's known that ATP depletion in cells can reduce the cells' insulin sensitivities (http://scholar.google.com/scholar?hl=en&q=intracellular+ATP+insulin+sensitivity).

As far as that case report goes, however, it's also worth noting that there are many old articles describing a higher frequency of cavities ("dental caries") in the context of phosphate depletion, and there are old articles describing the "anticariogenic" effects of an adequate phosphate intake (within the normal range of dietary intakes) (http://scholar.google.com/scholar?hl=en&q=sodium+phosphate+caries+OR+anticariogenic+OR+cariogenic). On the one hand, that's not surprising. Everyone knows that hydroxyapatite contains phosphate, etc. But, in the vast majority of the research that comes out these days, there's this overriding assumption that dietary phosphate is bad for bones and is going to cause calcium to be lost from the bones, etc. Obviously, I think one should be careful with sources of phosphate and not take large amounts of any source of phosphate, from food or otherwise, at any one time, so as to allow the kidneys to filter it and to allow the phosphate to be transported into cells. But the information on the utilizable phosphate content of many vegetable/plant-based foods is probably very inaccurate, in my opinion. Phytates may provide very, very little utilizable phosphate, as discussed in past postings, but these are just my opinions.

Interactions of Phosphate Metabolism With Energy Metabolism and Adenosine Metabolism

These are some articles showing that phosphate availability can be an important factor that determines the rates of salvage of purine nucleotides and nucleosides, the adenylate charge, and the rate of deamination of adenosine to inosine [Matsumoto et al., 1979: (http://www.jbc.org/cgi/reprint/254/18/8956.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/479172); Lockett et al., 1995: (http://www.ncbi.nlm.nih.gov/pubmed/8579734); (http://scholar.google.com/scholar?hl=en&q=energy+%22inorganic+phosphate%22+salvage+purine+OR+adenylate)]. Matsumoto et al. (1979) discussed the fact that inorganic phosphate [Pi, or PO4(3-)] normally inhibits adenosine monophosphate (AMP) deaminase activity, thereby preventing the catabolism of adenosine to inosine. This catabolism, however, can serve to maintain the energy charge, paradoxically, during the inhibition of energy metabolism (Matsumoto et al., 1979). But even when ATP levels are being maintained "normally," the sequestration or loss of intracellular phosphate tends to lead to the loss of adenosine nucleotides (reference 6, cited in 1979). Maj et al. (2000) found that the adenosine-induced inhibition of adenosine kinase (AK) activity, which is a major purine salvage enzyme in the brain and other tissues, decreases as the inorganic phosphate concentration increases. AK is sometimes viewed as being "bad" in the context of cerebral ischemia, and AK inhibitors can reduce brain damage due to ischemia by maintaining adenosine availability, etc. That's another reason that the provision of phosphate in the form of ATP disodium or another purine nucleotide might be advantageous. Phosphate depletion tends to produce a loss of adenosine (and, by extension, guanosine) nucleotides, and phosphate supplementation could have a mixture of beneficial and less-than-beneficial effects, particularly in the short term, on purine metabolism in the brain, for example. Increasing AK activity (meaning the phosphorylation of adenosine) without providing more exogenous adenosine could tend to decrease adenosine availability for cerebral blood flow autoregulation [Sciotti and Van Wylen, 1993: (http://www.ncbi.nlm.nih.gov/pubmed/8436611); (http://scholar.google.com/scholar?hl=en&q=%22adenosine+kinase%22+brain). It's partly because the concentrations of adenosine, both intracellularly (and extracellularly), are normally far lower than the Km of AK for adenosine. The same argument could be made in the case of the phosphate-mediated inhibition of AMP deaminase. Under conditions of low-level ischemia, as in a person with ATP depletion or purine nucleotide depletion (because of repeated cycles of ischemia or pronounced activation of the noradrenergic stress-response system in the brain), the degradation of AMP to IMP can, paradoxically, be "good," up to a point. In any case, there can even be strange short-term effects, in my opinion, of ATP disodium that could be explained, in part, by those paradoxical aspects of adenosine metabolism. Additionally, some few days may be required for changes in A1 adenosine receptor density or sensitivity to occur, even though extracellular adenosine levels are generally kept almost constant (in part by A1 adenosine receptor activation) [Andresen et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10490889)]. For example, A1 adenosine receptor antagonists tend to increase extracellular adenosine (Andresen et al., 1999) and can also have mood elevating effects or the like. Although the steady-state levels of extracellular adenosine and the sensitivities of adenosine receptors will generally adapt efficiently, in my opinion, to changes in stimulus-evoked increases in extracellular adenosine (exogenous ATP would be expected to primarily or almost exclusively augment stimulus-evoked extracellular adenosine concentrations and not steady-state extracellular adenosine levels), those adaptations could, in my opinion, require a day or two to take place. In my experience, there was some kind of threshold dosage, in the short term, above which there were no transient periods of somnolence or the like. I don't even know how I'd describe that type of thing, but my point is that there's some steady state that's reached and that there could be, in my opinion, potential for complex interactions with phosphate homeostasis. And the other point was that the use of sodium phosphate could disturb adenosine metabolism in the short term (and potentially the long term), even if one could not say that the effects are exclusively "bad."

Cholesterol in Steroid Hormone Biosynthesis; Cholesterol (and Vitamin D) in Hedgehog Signalling in the Brain and Liver

This is a great article [Kanat et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17234355)] that I cited in a recent posting (http://hardcorephysiologyfun.blogspot.com/2009/06/provocative-articles-showing-effects-of.html), and the authors discuss evidence that some cholesterol-lowering treatments may reduce the serum concentrations of testosterone, cortisol, or other adrenal steroid hormones. I remember seeing case reports of decreases in testosterone from some cholesterol-lowering approaches, but it's easy to "forget" that type of thing. I was just reading about the effects of squalene, a supplement that, in my opinion, doesn't sound very useful, on testosterone levels in animals, but, for some reason, that didn't remind me of the related avenues of research. Kanat et al. (2007) also mentioned that researchers had generally not found any increases in serum luteinizing hormone (LH) or follicle-stimulating hormone (FSH) in response to the decreases in serum testosterone. That's not that surprising, and I think that some people who write physiology textbooks or whatever other traditional reference "manuals" have this idea that the endocrinological regulatory mechanisms really work efficiently and "strictly" or "tightly" regulate the concentrations and cellular responses of hormones. It's hard for me to understand that, given the thousands of case studies and articles showing that failures in regulatory mechanisms are commonplace. That's a separate issue, though.

That article by Kanat et al. (2007) is disturbing to see, and it goes without saying that those reductions in cortisol and testosterone could conceivably also occur in people who have very low cholesterol levels and are not taking cholesterol-lowering drugs, in my opinion. In a person taking no medications, one would not expect to see pronounced inhibition of de novo cholesterol formation, such as can be produced by some cholesterol by the statins, in extrahepatic cells. But I wouldn't be surprised to see some of those changes in adrenal or gonadal steroid hormones showing up in people who have pathologically-low cholesterol levels. But that doesn't mean that increasing serum cholesterol is likely to just neatly fix endocrinological abnormalities, especially in a person who has extremely low serum cholesterol levels and is suicidally depressed, etc. So someone would obviously want to talk with his or her doctor about these issues. There are countless other possible causes of endocrinological abnormalities that have little to do with cholesterol.

There's quite a bit of interesting research showing that dietary cholesterol can improve some forms of liver disease in animal models, and there's this researcher who suggested that cholesterol supplementation could be used to treat liver disease (http://www.dukehealth.org/HealthLibrary/News/10021):

"Li pointed out that the findings could have other theoretical implications as well. He said giving alcoholics supplemental cholesterol could help slow down or prevent the occurrence of alcoholic liver disease, even chronic alcoholic induced cirrhosis, characterized by replacement of liver tissue by scar tissue, leading to progressive loss of liver function."

It's important to note that a lot of research shows that very high dietary cholesterol intakes in animals worsens the fatty liver disease that results from the animals' consumption of large amounts of dietary fat, etc. But many of the articles showing apparently therapeutic effects of cholesterol use much lower dosages than researchers have used in many of the dime-a-dozen articles on the effects of dietary cholesterol. I looked up some of the research that Li has been involved in, and it's interesting. He and his colleagues are researching some of the cell groups and signalling pathways, such as in relation to hedgehog signalling (by proteins such as sonic hedgehog (SHH), indian hedgehog, and desert hedgehog), that influence liver regeneration or repair, even in adult mammals [(http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=Yin-Xiong+Li+cholesterol); (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=Yin-Xiong+Li+fetal+alcohol); (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=Yin-Xiong+Li+cholesterol+OR+hepatic+OR+hepatocyte+OR+hedgehog)]. SHH is important for brain development, and deletion of either megalin, an endocytic receptor that also transports vitamin D, bound to vitamin D receptor, and is also a receptor for SHH (among other ligands), or patched, the classical SHH receptor, produces holoprosencephaly (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=holoprosencephaly+megalin+OR+patched). In that type of profoundly disordered brain development, the brain doesn't develop two distinct hemispheres, etc.

A few years ago, I was thinking that vitamin D3 depletion during brain development could disturb hegehog signalling and contribute to developmental brain disorders that have been suggested to be associated with vitamin D depletion. It was basically like applying the findings of Bijlsma et al. (2008) [Bijlsma et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/17870251)] to brain development, but I think it's probably difficult to tell what, if any, effects on hedgehog signalling would result from vitamin D depletion (or if the effect would be significant, etc.). It would be very complex. Those authors, though, have done research showing that 7-dehydrocholesterol inhibits SHH signalling by substituting for cholesterol in hedgehog processing. Vitamin D or 25-hydroxyvitamin D or hormonal vitamin D could conceivably also substitute for cholesterol, because vitamin D steroids exist as rotamers and convert back and forth between s-cis and s-trans rotamers hundreds of times per second [(http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=%22vitamin+D%22+%22s-cis%22+OR+%22s-trans%22); (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=%22vitamin+D%22+rotamer+%22s-cis%22+OR+%22s-trans%22); (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=%22vitamin+D%22+%22cholesterol-like%22+%22s-cis%22+OR+%22s-trans%22)]. So vitamin D and its metabolites can assume cholesterol-like conformations, etc. Maybe someone's already shown that vitamin D or its hydroxylated metabolites can't substitute for cholesterol in hedgehog processing, but anyway--it's just one of my wild, old ideas that sounds far-fetched to me now. I briefly looked at all of these byzantine interactions of VDR-ligand-mediated changes in gene expression with SHH signalling, etc., based partly on the fact that megalin is a receptor for both vitamin D receptor (vitamin-D-bound or drug-ligand-bound) and SHH. I don't even remember what effect I thought vitamin D repletion would have on SHH signalling. The mechanisms that Bijlsma et al. (2008) have researched look focused to me and look to be more plausible than the potential interactions with vitamin D metabolism and signalling appear to be. It looks like a nightmare (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=hedgehog+cholesterol+%22vitamin+D%22+OR+hydroxyvitamin+OR+dihydroxyvitamin), partly because VDR activation can influence the expression of SHH and other hedgehog proteins and can also influence the responsiveness to the cellular actions of SHH, etc. There might be some capacity for vitamin D or its metabolites to serve as substrates for enzymes that metabolize or otherwise utilize cholesterol or for proteins that bind cholesterol, etc. Some orally-administered vitamin D3 binds to lipoproteins [this article is probably not the best, as far as describing that, but it shows the effect: Teramoto et al., 1995: (http://www.ncbi.nlm.nih.gov/pubmed/7575591)], and that occurs more with oral vitamin D than with vitamin D from the skin (which circulates, almost exclusively, bound to VDR, supposedly). That type of binding is not necessarily indicative of cholesterol-mimesis, but it's interesting.

In any case, I don't completely understand the research by Li and colleagues yet, but the covalent binding of cholesterol to SHH is necessary for SHH to be completely functional [this is one paper that Li is a coauthor of: Sicklick et al., 2005: (http://www.nature.com/labinvest/journal/v85/n11/full/3700349a.html)(http://www.ncbi.nlm.nih.gov/pubmed/16170335)]. The authors suggest that some cholesterol-lowering drugs may reduce hepatic stellate cell activation (mitogenic activation, proliferation, etc.) and liver collagen accumulation by reducing SHH signalling (i.e. by reducing cholesterol availability for binding to SHH). That's an interesting hypothesis, but I can't find any mention of supplemental cholesterol in these articles I've looked at so far. In another article that Li is a coauthor of [Yang et al., 2008: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2196213)(http://www.ncbi.nlm.nih.gov/pubmed/18022723)], the authors basically say that SHH is upregulated in hepatic stellate cells, in response to their activation with platelet-derived growth factor (PDGF) and other mitogens, etc., and helps to promote the survival of those stellate cells. That's evidently viewed as being pathological. So how, in that type of context, would dietary cholesterol supplementation ameliorate liver disease? It sounds like the activation of hepatic stellate cells is kind of a mixed bag and may be able to produce either regeneration or an exacerbation of fibrosis, in my opinion. But I'm not all that sure about that.

I've never seen anyone suggest that, though, about increases in intracellular cholesterol levels in cells in the liver being able to augment cell proliferation or survival, potentially, by maintaining the functionality of SHH and other hedgehog proteins. It's completely new. That might be relevant to research on Alzheimer's disease or psychiatric conditions [(http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=cholesterol+hedgehog+Alzheimer%27s); (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=antidepressant+hedgehog)]. A lot of the research on cholesterol metabolism in relation to Alzheimer's disease is still focusing on isoprenoid signalling, and a lot of it doesn't seem to be going much of anywhere, in my opinion. If hedgehog signalling is involved in progenitor cell survival in the liver in adult mammals [Sicklick et al., 2006: (http://ajpgi.physiology.org/cgi/content/full/290/5/G859)(http://www.ncbi.nlm.nih.gov/pubmed/16322088)], then maybe it also could promote neuronal progenitor cell survival. I don't know if decreases in neuronal progenitor cell viability do, in fact, contribute to psychiatric or neurodegenerative diseases. There's a lot of research suggesting that they do, but it's always seemed sort of unclear, in my mind, what the real mechanism would be. Exercise and a grab bag of other factors increases neuronal progenitor cell proliferation in the subventricular zone, etc., but most of the cells don't survive. There's a patent on using a "hedgehog agonist to treat depression" and some articles on hedgehog signalling in neuronal progenitor cell proliferation (hippocampal neurogenesis) [(http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=antidepressant+hedgehog); (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=psychiatric+sonic+hedgehog+adult)]. When one takes a very crude look at things, cholesterol is like a weak hedgehog agonist, in my opinion, because it activates or maintains hedgehog functionality. This posting is deteriorating. A lot of those, in that last search, look to be related to the proliferation of progenitor cell populations, but hedgehog could also conceivably promote or regulate the cell-cycle re-entry of terminally-differentiated neurons (this is "bad" cell-cycle re-entry) (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=Alzheimer%27s+hedgehog+%22cell+cycle%22+reentry+OR+%22re-entry%22).

In any case, I was going to mention that there's obviously another side to liver-related issues and some cholesterol-lowering drugs, and this is one article that addresses some of those issues and concerns [Argo et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18666246)]. There are two sides to lots of those issues, and I'm not going to get into a discussion about all of that.

Potential Problems Associated With Excessive Zinc and Copper Supplementation

These are some more articles that discuss all of the mechanisms by which an excess of intracellular, free zinc can cause mitochondrial dysfunction and toxic effects on many other cellular processes. The authors of this article [Lemire et al., 2008: (http://oldwebsite.laurentian.ca/chem/vappanna/publications/J.%20Applied%20Toxicology%202008.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/17582580)] discuss the fact that an excess of zinc can inhibit various TCA cycle enzymes, such as aconitase, and other mitochondrial enzymes. Although the authors note that zinc can interfere in a generalized way with enzymes and proteins that contain iron-sulfur clusters, aconitase is known to also utilize nonheme iron (I mean nonheme iron that is also non-iron-sulfur-cluster-bound iron) [see, for example, Lee et al., 1996: (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=aconitase+nonheme+iron)]. Zinc can be utilized to form zinc protoporphyrin, and I wonder if zinc isn't actually incorporated into iron-sulfur clusters. But it wouldn't have to be, and zinc could reasonably be expected to displace, with particular ease, nonheme iron (i.e. Fe2+/Fe3+) from its binding sites on enzymes whose catalytic activities or regulatory functions depend on the presence of bound, nonheme iron.

I think neurotoxicity from excessive zinc supplementation is a very serious issue, and, in my opinion, low-level, pathological changes, such as psychiatric symptoms or gradually-progressing neurotoxicity, may result from dosages of zinc that many people would not view as being especially massive. Only a relatively few authors, as far as I can tell, have written articles, over the last few decades, discussing the potential hazards of zinc supplementation at the more commonly-used dosages. Even fewer articles on the nutritional aspects of zinc have considered that problems with zinc supplementation may have nothing to do with copper depletion, even when copper is depleted as a result of the excess zinc. In my opinion, based on the literature, many of the cases of neurotoxicity associated with excessive zinc supplementation (there are many, many case reports in the literature, and I don't feel like listing dozens of them out, right now, in this posting) may have had relatively little to do with copper depletion, and one sees that copper supplementation, in many cases, did not very effectively ameliorate the neurological disorders and demyelination that researchers had found in association with excessive intakes of zinc. [Some of the articles and case reports are scattered throughout the results of this search: (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22zinc+supplementation%22+neurotoxic+OR+neurological+OR+demyelinating+OR+demyelination+OR+%22white+matter%22), and this is an "instructive-but-not-comprehensive" list of articles that google scholar classifies as being related to an article on excessive zinc supplementation: (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=related:-bvm-ASLfasJ:scholar.google.com/)]. In most cases, the researchers have discontinued the zinc supplementation at the same times they have initiated copper supplementation, and then the researchers have attributed the modest improvements in the neurological conditions or anemia or thrombocytopenia or pancytopenia to the copper repletion. In reality, the absence of zinc may have been the primary and more important factor that led to the improvements.

I've discussed, in past postings, the extraordinarily complex aspects of zinc metabolism and homeostasis, and another issue is that serum copper and ceruloplasmin are not sensitive or very reliable indicators, in my opinion, of the intracellular copper concentrations or of copper status in people who are not grossly copper-deficient. Serum zinc is also not thought to be a reliable measure of zinc status [Fung et al., 2002: (http://www.ncbi.nlm.nih.gov/pubmed/16215013)], and Fung et al. (2002) found that, even in people who had evidently been deficient in zinc and who had appeared to respond favorably to zinc supplementation, zinc supplementation did not increase serum zinc. These are major obstacles to nutritional research on zinc, in my opinion, and I would seriously question the validities of many descriptions, in the literature, of "zinc deficiency." Here's an article whose author discusses the potential neurotoxicity of zinc supplementation [Levenson, 2005: (http://www.ncbi.nlm.nih.gov/pubmed/15869126)], and here's a remarkable article, from 1989, in which Fosmire (1989) [Fosmire, 1989: (http://journals.lww.com/nutritiontodayonline/pages/articleviewer.aspx?year=1989&issue=05000&article=00005&type=abstract)] displayed remarkable prescience and subjective insight in relation to the potential problems that, in my opinion, could develop with dosages of zinc that many people would not view as being "excessive." I don't think zinc supplementation is a good idea in most cases, but that's just my opinion. I also don't think copper supplementation, above some tiny dosage (such as ~250-500 micrograms of elemental copper, to reach the RDA in combination with one's specific dietary intake), is a good idea, in many cases, but that's also just my opinion. A person would obviously want to discuss these issues with his or her doctor, given that one's unique, individualized nutritional needs are of paramount importance. I also think that the use of supplemental copper to compensate for the copper-depleting effects of zinc supplementation does not make sense and generally just has the potential to cause copper toxicity, but these are just my opinions.

I know this is a disturbing topic, but there's a kind of insanity in the way the different branches of research on zinc remain separated from one another. There's a vast amount of research showing copper-independent neurotoxic effects and extraordinarily complex, dynamic mechanisms of neurotoxicity from excesses of free zinc (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=zinc+neurotoxicity), and then there's the nutritional research on zinc. People seem to think that zinc-induced neurotoxicity is an all-or-nothing phenomenon. The assumption is that high dosages, such as are discussed in the case reports of neurotoxicity or demyelination, can cause problems but that the absence of overt neurological symptoms, in people taking lower doses, is in some way evidence that those lower dosages are not producing any pathological effects in the brain and spinal cord. It makes no sense to me to think that there would be no potential for problems in response to some of these lower dosage ranges, but that's just my opinion. The main reason I think that is that a multitude of factors can influence the amounts of zinc that are being released from intracellular binding sites, and I would expect that both the amounts of intracellular free zinc in neurons and the consequences of that zinc could be drastically different among different individuals with similar serum zinc levels and zinc intakes. The authors of this article discuss the possibility that the therapeutic intake range for zinc may well be small [Maret and Sandstead, 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16632171)], and it's time to face some of these issues, which I feel are serious, relating to zinc and copper supplementation.

Acyl-CoA Accumulation, Ketogenic Substrates, Fatty Liver Disease, Moderation, and Sustainability

These articles [Aiello et al., 1983: (http://jds.fass.org/cgi/reprint/67/8/1707.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/6480960); Henning and Hird, 1972: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1174516&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/4664932)] show that butyrate, such as from the highly-soluble sodium butyrate, is an effective ketogenic substrate in hepatocytes (in the livers of cows) (Aiello et al., 1983) or in the epithelial cells of parts of the GI tract in rabbits (Henning and Hird, 1972). There's nothing that's really magical about a ketogenic compound, and even glucose is obviously an indirect, ketogenic substrate. But the issues of interest are the efficiencies with which different compounds can be converted into ketones and the extents to which increases in the availabilities of different compounds can increase ketone formation or oxidation, etc. Increasing glucose in one's diet is unlikely to increase ketone formation very efficiently, past a certain point, for example.

I should mention that the accumulation of short-chain or branched-chain or long-chain or any other types of acyl-CoA thioesters can potentially contribute to fatty liver disease in the long term or under other circumstances, in different individuals. That's my opinion, and acyl-CoAs just inhibit all sorts of different mitochondrial enzymes and inhibit ATP production, etc., and are thought to contribute to the development of fatty liver disease (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=acyl-CoA+%22fatty+liver%22+mitochondrial). I've discussed this in past postings. Although there is research showing that high-fat diets can sometimes actually be beneficial to fatty liver disease, to the extent that high-protein, low-carbohydrate diets can reduce insulin levels, I find it hard to believe that most people would be able to sustain very high-fat, "ketogenic" diets. Increasing one's fat intake is not necessarily going to cause fatty liver disease, but the point is that cells in the liver and other parts of the bodies of adult humans (such as in astrocytes, in the brain) do not have as much of a capacity to oxidize fatty acids as the cells of children and adolescents do. Most of the research, in my opinion, shows that high fat diets, in combination with the carbohydrates that most people eat and that are almost impossible not to eat, do increase a person's risk of developing fatty liver disease. It's just something to be aware of, given that ketogenic substrates, such as HMB and butyrate, can cause the accumulation of acyl-CoAs that can become toxic, in my opinion.

I think there can be a tendency for people to think that they have to either commit to a full-blown, restrictive, "ketogenic diet" or to do nothing, but those are not the only approaches. I understand that carbohydrate ingestion tends to suppress ketone formation, but this is only true to a certain point, in my view. It's not an all-or-nothing phenomenon. Eating carbohydrates doesn't shut down ketone formation completely, and one doesn't, in my opinion, need to eat 100 grams a day of liquid fats to slightly increase ketone formation by astrocytes or by the liver or intestinal epithelial cells. One approach would be to, of course, talk to one's doctor before taking anything and to discuss the possibility of taking small doses of some of these ketogenic substrates. Researchers are discussing the potential usefulness of an increase in ketone availability in many different conditions, including Alzheimer's disease and psychiatric conditions, etc. But combining small doses of some of these ketogenic compounds with something like resistance exercise, which tends to increase ketone formation and utilization in a more subtle and physiologically-sustainable way, seems, in my opinion, to be a more rational approach than does the use of one of these drastic, high-dose-or-nothing, dietary interventions.

Ketogenic Amino Acids and the Regulation of Cholesterol Biosynthesis

The authors of this article [Noda and Ichihara, 1976: (http://www.ncbi.nlm.nih.gov/pubmed/1002682)] discuss research showing that the oxidation of ketogenic amino acids to CO2 (under conditions in which they are completely oxidized), such as tyrosine and leucine, provides twice as much ATP as the oxidation of gluconeogenic amino acids. The authors refer to a book, as the cited reference for that statement, and so I'm not sure what they actually mean. I'm assuming they mean that the oxidation of the carbons of acetyl-CoA, derived from the oxidation of the ketones that contain the leucine or tyrosine carbons, in the tricarboxylic acid cycle can serve to generate twice as much net ATP as the oxidation of the amino-acid-derived carbons in glucose formed by gluconeogenesis from amino acids.

It's interesting that the cytosolic enzyme 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) is one of two enzymes that forms HMB, a lipogenic/ketogenic leucine metabolite discussed in previous postings, and is also a key enzyme involved in the oxidation of tyrosine to acetoacetate (ketone formation from tyrosine) [reference 65, p. 728, discussed in: Schofield and Zhang, 1999: (http://alpha.life.nthu.edu.tw/~d888206/Pdf%20papers/2-OXO.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/10607676)]. 4-HPPD is sometimes referred to, in articles about HMB, as "KIC dioxygenase," or alpha-ketoisocaproate (KIC) dioxygenase, but it's more appropriate to say that 4-HPPD has KIC dioxygenase activity, in addition to its usual role of catalyzing the formation of homogentisate, an intermediate in tyrosine catabolism, from 4-hydroxyphenylpyruvate (Schofield and Zhang, 1999). Looking at the factors regulating that enzyme might be a starting point for understanding the mechanisms by which exogenous HMB could reduce leucine catabolism slightly (an effect that has been shown to occur, recently, in an animal study) etc. HMB can also be formed from isovaleryl-CoA, as discussed previously, by crotonase, which is more commonly known as enoyl-CoA hydratase [Rodriguez et al., 2004: (http://www.jbc.org/cgi/content/full/279/6/4578)(http://www.ncbi.nlm.nih.gov/pubmed/14612443?dopt=Abstract)]. Rodriguez et al. (2004) also discuss the fact that HMG-CoA can be converted into various isoprenoids (geranyl-CoA, etc.), which are intermediates in cholesterol biosynthesis, and then recycled back into 3-methylcrotonyl-CoA and HMB-CoA, by the enzymes of the so-called "mevalonate shunt." The authors also mention Smith-Lemli-Opitz syndrome, which is a genetic disorder that causes pathologically low plasma cholesterol levels and psychiatric symptoms. The disorder prevents the conversion of delta7-dehydrocholesterol into cholesterol, because of loss-of-function mutations in 7-DHC reductase, and causes toxic cholesterol precursors to accumulate and cause brain damage, etc. (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=7-dehydrocholesterol+Smith+Lemli+Opitz). Rodriguez et al. (1999) also mention that plasma 3-methylglutaconic acid can increase in people who have that genetic disorder, and researchers could investigate the possibility that the accumulation of intermediates in cholesterol biosynthesis or in the mevalonate pathway contribute to psychiatric symptoms associated with low plasma cholesterol. If that were the case, one might expect HMB or increases in ketone availability from other ketone precursors (other ketogenic substrates, other than HMB) to transiently worsen psychiatric symptoms but ultimately increase cholesterol formation and improve psychiatric symptoms, given that the normalized pool of cholesterol would be able to exert feedback inhibition of HMG-CoA reductase activity and prevent the accumulation of the intermediates. But these are just my opinions and avenues of thought in this area.

Neuroprotective and Supposed Antidepressant-Like Effects of Sodium Butyrate: Relevance to HMB Research and Energy Metabolism

A lot of these articles showing that butyrate (usually administered or used in vitro as sodium butyrate, or SB), a short-chain fatty acid similar in structure to HMB (3-hydroxy-3-methylbutyrate or 3-hydroxyisovalerate, discussed in the two previous postings), reduces the degradation of numerous proteins by proteasomes are relevant to research on HMB. There are many similarities among the effects of butyrate and HMB. HMB is thought to exert its anticatabolic effects by inhibiting proteasomal activity (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=proteasome+methylbutyrate+OR+%223-hydroxyisovalerate%22) and also by acting as a precursor of HMG-CoA and of cholesterol. The extent to which an HMB-induced increase in cholesterol formation contributes to the HMB-induced inhibition of proteasomal activity is unknown. SB is a nonselective inhibitor of histone deacetylase enzymes in vitro, and its histone deacetylase inhibitory effect, at least in vitro, is thought to contribute to its inhibition of TNF-alpha-induced NFkappaB (NFkB) transcription factor [a.k.a. the "Rel" family of subunits that form the dimers that comprise NFkB transcription factors: (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=RelA+RelB)] activation in the cytosol (Yin et al., 2001: (http://www.jbc.org/cgi/reprint/276/48/44641)(http://www.ncbi.nlm.nih.gov/pubmed/11572859?dopt=Abstract); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22sodium+butyrate%22+proteasome)]. Butyrate doesn't prevent the ubiquitination of IkB proteins (inhibitors of NFkB activation) but causes them to acccumulate as ubiquitin-conjugated proteins, without being degraded in proteasomes, evidently (Yin et al., 2001). HMB is also thought to exert anti-inflammatory effects by suppressing NFkB activation, as a result of the HMB-induced suppression of "proteasomal activity" [Baxter et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16006030)].

I don't doubt that some of those mechanisms are important, and a decrease in the activation of NFkB transcription factors can be antiproliferative and can downregulate the expression of numerous pro-inflammatory cytokines (cytokines that suppress mitochondrial functioning), etc., but SB is produced by microorganisms in the GI tract and is known to be the major energy substrate for colonocytes in the submucosal layers (I forget the terminology) of the colon (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22sodium+butyrate%22+energy). The in vitro research probably uses bizarre conditions and shows that SB can induce apoptosis of colon cancer cells. It looks like SB is pro-apoptotic at high but not low concentrations (0.5 mM to 2 mM) [Singh et al., 1997: (http://carcin.oxfordjournals.org/cgi/reprint/18/6/1265.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/9214612)]. But SB supposedly doesn't produce very strong histone deacetylase inhibition in the brain in vivo in animals, but it does produce neuroprotective effects in all sorts of different models (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22sodium+butyrate%22+neurodegenerative+OR+neurological+OR+neuroprotective+OR+Parkinson%27s+OR+Huntington%27s+OR+ischemia+OR+ischaemia+OR+hypoxia+OR+anoxia). Sodium butyrate has also produced some "antidepressant-like" effects in animal models of depression (http://scholar.google.com/scholar?q=%22sodium+butyrate%22+antidepressant&hl=en&lr=). Sodium butyrate is also sold as a supplement (http://www.google.com/products?q=sodium+butyrate&hl=en&aq=f).

Anyway, I just put this information up here, but I have no idea what the dosage range would be. One would obviously want to discuss this type of thing with one's doctor, and the most obvious, potential problem would be the disturbances in phosphate or calcium homeostasis in response to something like this. The infusion of 3-hydroxybutyrate, a "ketone" that doesn't have a carbonyl group but is defined as being a ketone, and acetate, for example, can increase plasma bicarbonate, and this effect appears to be the result of the metabolism of the organic acids/fatty acids and not from effects on phosphate homeostasis, in some articles. But these organic anions can just have strange effects, and it's something to be aware. Many medications can affect acid-base homeostasis and could interact with sodium butyrate or HMB. Some anticonvulsants act as carbonic anhydrase inhibitors, for example, and could interact with these types of short-chain fatty acids (such as sodium butyrate) or branched-chain organic acids/fatty acids (such as HMB).

In my opinion, sodium butyrate probably acts mostly as an energy substrate, but that doesn't exclude other mechanisms. I also think the research on sodium butyrate is likely to be relevant to future research on the mechanisms of action of HMB. Both compounds inhibit proteasomal activity and may have overlapping or similar effects, but I don't think it's going to be as simple as testing HMB as a "histone deacetylase inhibitor." Histone acetylation is extraordinarily complex and dynamic, and to think that one can treat a multitude of conditions with histone deacetylase inhibitors is not realistic, in my opinion. Vitamin D receptor activation can increase histone acetylation (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22vitamin+D%22+histone+acetyltransferase+OR+acetylation), much as sodium butyrate supposedly does (histone deacetylase inhibition leads to increases in the acetylation of histone proteins). But it's clear, in my opinion, that a lot of the effects of sodium butyrate cannot be explained in terms of histone acetylation.

Notes on HMB; Leucine as a Supposedly-Ketogenic Amino Acid

This article [Kuhara et al., 1982: (http://www.ncbi.nlm.nih.gov/pubmed/7116632)] describes leucine as being a "potent ketogenic amino acid," and I guess there can be a significant contribution of leucine-derived branched chain organic acids, such as HMB/3-hydroxyisovalerate, to ketogenesis. About 5 percent of leucine normally is converted into HMB, apparently. The only other thing I thought of is that the slight elevation in plasma branched-chain amino acids (Holocek et al., 2009) [Holecek et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19056452)] might conceivably produce tryptophan (TRP) and tyrosine (TYR) and phenylalanine (PHE) depletion from the brain, in my opinion, but it looks like the effect is probably not even as pronounced as the increase in the plasma BCAA (leucine+isoleucine+valine)/(TYR+TRP+PHE) ratio from a high-protein meal. In dietary protein, 20-30 percent of the amino acids are BCAA's, if memory serves, and this causes the plasma BCAA/(TYR+TRP+PHE) ratio to increase progressively as the dietary protein intake increases [Fernstrom et al., 1979: (http://www.ajcn.org/cgi/reprint/32/9/1912.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/573061)]. There's the potential that that effect, to the extent that it could occur in response to the mild, apparent "leucine-sparing" effect of HMB (Holocek et al., 2009), could, in my opinion, produce transient worsening of mood or other psychiatric complications in people. Although some of those trials with HMB suggest that the opposite effect would occur, I can't really put a lot of faith in some of these articles on the effects of BCAAs on the brain. They're used as neuroprotectives and as a treatment for mania (leucine and other BCAAs), and I doubt HMB would have the same effects. It's not an amino acid and wouldn't be expected to compete with tyrosine, tryptophan, and phenylalanine for entry into the brain. But I can't be sure about that, and it's obviously something a person would want to discuss with one's doctor. It's possible that the supposed neuroprotective effects of leucine and BCAAs, as in spinocerebellar degeneration, etc., are not mediated by glutamine or 2-oxoglutarate sparing or whatever mechanism has been suggested but are the result of a ketogenic effect of leucine in astrocytes (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=BCAA+spinocerebellar). The authors of those BCAA-as-neuroprotective articles view the BCAAs as being energy substrates or as being capable of decreasing glutamate, though, I think. I don't know what the proposed mechanisms are, besides those mechanisms. HMB can also act as a precursor of fatty acids and could conceivably cause problems in people with liver disease, but those old articles show that it's, evidently, preferentially incorporated into cholesterol. I discussed the BCAA issues in a past posting (http://hardcorephysiologyfun.blogspot.com/2009/02/potential-psychiatric-pitfalls-in.html).

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)].

Epstein-Barr Virus Infection of Astrocytes and Monocytes: Potential Relevance to Research on Multiple Sclerosis and Astrocyte Cell Cycle Re-Entry

These articles [Chaudhuri, 2005: (http://www.ncbi.nlm.nih.gov/pubmed/15617877); Behan et al., 2002: (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=Behan+Chaudhuri+Roep+%22THE+PATHOGENESIS+OF+MULTIPLE+SCLEROSIS+REVISITED%22); VanAmerongen et al., 2004: (http://www.direct-ms.org/pdf/VitDMS/VanAmerongenVitDMSreview.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/15054436); Cepok et al., 2005: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1077174)(http://www.ncbi.nlm.nih.gov/pubmed/15841210); Diesel et al., 2005: (http://clincancerres.aacrjournals.org/cgi/content/full/11/15/5370)(http://www.ncbi.nlm.nih.gov/pubmed/16061850); Sanders et al., 1996: (http://www.ncbi.nlm.nih.gov/pubmed/8799216); Prokova et al., 2002: (http://www.jbc.org/cgi/content/full/277/11/9342)(http://www.ncbi.nlm.nih.gov/pubmed/11781310?dopt=Abstract); Koch et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/17050217)] are really good, and Chaudhuri (2005) suggests that vitamin D repletion during brain development may protect against abnormal astrocyte apoptosis later in life and thereby confer protection against multiple sclerosis. This is interesting and is similar to the vitamin D hypothesis of schizophrenia [McGrath and colleagues: (http://scholar.google.com/scholar?q=%22vitamin+D%22+schizophrenia&hl=en&lr=)], in the sense that there's this concept of vitamin D deficiency, during development, creating abnormalities in brain development that do not manifest themselves until relatively later in life than one might expect them to. For example, vitamin D depletion during brain development drastically decreases the expression and protein content of the low-affinity neurotrophin receptor (p75NTR), which binds all of the neurotrophins and plays crucial roles in the regulation of not only apoptosis or protection against apoptosis, by NGF and other neurotrophins (NT-3, NT-4, BDNF, etc.), but in the trophic effects of NGF in the adult brain.

Holmoy (2008) suggested that vitamin D repletion could protect against brain damage due to late Epstein-Barr Virus (EBV) infection (i.e. after early childhood, when infection is often asymptomatic or less destructive to the brain), which tends to produce an expansion of autoreactive T-cell populations [Holmoy, 2008: (http://www.ncbi.nlm.nih.gov/pubmed/17574770)]. There's actually research showing that EBV can infect astrocytes [Menet et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10438862)] and monocytes and other cells of the monocyte-macrophage lineage [Savard et al., 2000: (http://www.ncbi.nlm.nih.gov/pubmed/10684275); (http://scholar.google.com/scholar?num=100&hl=en&lr=&cites=16392233215499070431)], which means that EBV may very well infect microglia and perivascular macrophages, etc. There still seems to be a popular sentiment that EBV only infects B-cells and epithelial cells, but there is overwhelming evidence that this is not the case and that EBV infects cells in the brain en masse during infectious mononucleosis (the term mononucleosis refers to the characteristic finding of monouclear phagocyte, or monocyte, infiltration of tissues infected by EBV; most cases of infectious mono are the result of primary EBV infection, although some can be from primary CMV infection or EBV infection that causes polyclonal, EBV-infected B-cells to start producing anti-CMV IgM and make it look like a person who had previously been infected with CMV has a primary CMV infection). I don't feel like going through papers and discussing them, but here are some hastily-done searches showing vast numbers of articles on the subject (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=mononucleosis+brain+OR+encephalitis+OR+encephalopathy+OR+meningitis+OR+meningeoencephalitis); (http://scholar.google.com/scholar?q=mononucleosis++brain+OR+encephalitis+OR+encephalopathy+OR+meningitis+OR+meningeoencephalitis&num=100&hl=en&lr=&scoring=r&as_ylo=2004)]. The pro-inflammatory response during infectious mono is massive, and the notion that the blood-brain barrier would be impermeable to infiltration by EBV-infected, polyclonal B-cells is not reasonable. Also, an important distinguishing feature of infectious mono is enlargement or lymphadenopathy in the posterior cervical lymph nodes that provide lymphatic drainage to the brain, producing a stiff neck, etc. The oligoclonal IgG antibodies in the CSF of people with multiple sclerosis have repeatedly been shown to bind EBV proteins (Cepok et al., 2005), and Cepok et al. (2005) go into all the research showing that type of thing. It's possible that the immune response is being directed against other latently-infected B-cells, etc., but the evidence is pretty substatial that late EBV infection plays some role in the etiology of multiple sclerosis, in my opinion. To think that astrocytes and probably microglia and other cell types in the central nervous system would be spared infection makes no sense to me. So it probably occurs in many or most people who are infected with EBV (90-95 percent of the US population, by age 26-27), and one might look for some differences in the degree of ongoing damage or in the pattern of gene expression by EBV (i.e. the latency pattern) in astrocytes or microglia, etc. (discussed below) of people who go on to develop multiple sclerosis, in comparison to controls.

Behan et al. (2002) discuss a lot of evidence that inappropriate astrocytic cell-cycle re-entry plays a prominent role in the etiology of multiple sclerosis, and the authors, one of whom is Chaudhuri (see Chaudhuri, 2005), also discuss the association of multiple sclerosis with glioblastoma multiforme and with rare, diffuse forms of gliomas, etc. That article is superb and is really brilliant, and yet it's not even indexed in Medline. The fact that vitamin D analogs have been used to treat glioblastoma multiforme is interesting, and the effects of vitamin D receptor (VDR) ligands, including calcitriol itself, on the astrocytic cell cycle could suggest that they could protect against astrocytic cell cycle re-entry and apoptosis in people with multiple sclerosis. I tend to think they wouldn't be all that effective in that regard and that the focus of Chaudhuri (2005) on the developing brain makes more sense. But the focus on astrocytes (Chaudhuri, 2005; Behan et al., 2005) is really intriguing, and it suggests to me that other measures might protect against abnormal astrocyte proliferation and apoptosis (i.e. guanosine and other intravenously-administered purine nucleotides or those in combination with energy substrates, etc.). That's just my opinion. It's interesting that VDR activation leads to very complex interactions with the transforming growth factor-beta signalling cascade, such as by forming heterodimers with Smad3 and potentiating many Smad3-induced transcriptional changes (VanAmerongen et al., 2004), and that the EBV latent membrane protein-1 suppresses Smad3-dependent transcriptional changes (Prokova et al., 2002). Smad3 is phosphorylated by type I TGFbeta receptors and is thereby activated as a transcription factor. Smad3 interacts with many proteins, but the suppression by LMP1 of the TGFbeta-induced and Smad3-mediated increase in p21WAF1/Cip1 expression (Prokova et al., 2002) is a relatively specific intersection with the transcriptional program that tends to be induced by VDR activation. The p21WAF1/Cip1 gene is a major cell-cycle-regulatory gene whose expression is responsive to and increased by VDR activation. The gene product allows for enhanced DNA repair before cell division, etc., and contributes to the antiproliferative and differentiating effects of VDR activation. That's just one example, but it lends credence to the hypothesis of Holmoy (2008) and suggests that the interactions of VDR-ligand-induced transcriptional changes with EBV-induced transcriptional changes may be relatively direct and may go beyond the realm of VDR-ligand-induced increases in interleukin-10 output from monocytes, etc. It might be possible to look for the effects of vitamin D or its analogs on EBV-infected, cultured monocytes or astrocytes or to look for associations of 25-hydroxyvitamin D levels with the incidences of glioblastoma among patients with multiple sclerosis? That sounds pretty difficult. There are some recent articles discussing all the problems with detecting herpesviruses in the brain during autopsies. Serafini et al. (2007) [Serafini et al., 2007: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2118531)(http://www.ncbi.nlm.nih.gov/pubmed/17984305)] found that cells in perivascular regions of the brains of people with multiple sclerosis were immunoreactive for LMP1 and other latency-associated EBV proteins, and it doesn't sound like that can be casually attributed to infiltrating, EBV-infected B-cells, etc. Someone could look for an association between 25-hydroxyvitamin D levels at death and the latency pattern of EBV infection in the brains of people with MS (or just look for different latency patterns in people with MS). I'm just thinking out loud with this.