The Alanine-Glucose Cycle: Relevance to the Effects of Exercise on the Brain

The authors of this article [Roe and Mochel, 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16763896)] discussed the fact that skeletal-muscle myocytes normally export some alanine (ALN) and glutamine (GLN) and that the liver may utilize significant amounts of that ALN and GLN. Roe and Mochel (2006) referred to the process as being the "alanine cycle," and the process is also known as the "alanine-glucose cycle." Roe and Mochel (2006) noted that the ALN cycle is a "one-way street" that allows for alanine to be exported from the skeletal muscles and utilized by the liver as a precursor of pyruvate, particularly during strenuous exercise. Exercise is known to increase serum ALN, but that's obviously only one effect that occurs during some types of exercise. Evans et al. (2004) found that the intravenous infusion of L-ALN (that's not the same as beta-alanine, and beta-alanine supplementation can produce symptoms of neuropathy at relatively low dosages, in some cases [Harris et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16554972)]) improved some measures of cognitive functioning in nondiabetic people who were being made artificially hypoglycemic, but it's not clear if the effects were solely due to the entry of ALN into the brain or if the ALN-induced elevations in plasma lactate contributed to the effects [Evans et al., 2004: (http://www.ncbi.nlm.nih.gov/pubmed/15089788)]. Incidentally, oral GLN reliably elevates plasma ALN, and, as a representative article, Déchelotte et al. (1991) found that jejunally-administered GLN elevated plasma ALN in humans [Déchelotte et al., 1991: (http://www.ncbi.nlm.nih.gov/pubmed/1903599)].

That capacity of the skeletal muscles to export GLN and ALN is likely to be relevant to research on the effects of exercise on the brain. These articles don't show the effect as unequivocally as some other articles do [Dalsgaard et al., 2004: (http://jp.physoc.org/content/554/2/571.full)(http://www.ncbi.nlm.nih.gov/pubmed/14608005?dopt=Abstract); Kemppainen et al., 2005: (http://jp.physoc.org/content/568/1/323.full)(http://www.ncbi.nlm.nih.gov/pubmed/16037089?dopt=Abstract)], but the utilization of serum lactate during exercise allows the brain to decrease glucose utilization, and that means that the oxidation of lactate in astrocytes and neurons can exert a "glucose-sparing" effect. It's fairly clear to me that the utilization of lactate becomes more significant during high-intensity exercise than during low-intensity exercise, but the release of lactate from the muscles is also higher. Resistance exercise is likely to ultimately, in the long term, allow the muscles to export substantially more ALN and lactate and GLN than endurance exercise is, and I'm talking about the postexercise period and in the fasted state. Endurance exercise tends to not increase muscle mass (as in the percent lean body mass) very much, and an increase in the efficiency of something like the ALN-glucose cycle requires, in my opinion, some sort of actual increase in muscle tissue. I've discussed some of the details about resistance exercise in past postings.

More Details on Glutamine Metabolism in the Liver, With Reference to the Brain, and in the Brain, Also With Reference to the Brain

These are some articles [Iglesias et al., 2001: (http://cat.inist.fr/?aModele=afficheN&cpsidt=13399682); Jia et al., 2006: (http://wjg.wjgnet.com/downpdf.asp?url=/1007-9327/12/1373)(http://www.ncbi.nlm.nih.gov/pubmed/16552804); Schuster et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19324476); Hong et al., 1992: (http://www.ncbi.nlm.nih.gov/pubmed/1546897)] that show the capacity of glutamine (GLN) to protect against various types of liver damage in animals. GLN reduced the amount of damage produced by acetaminophen-induced liver failure in rats (Hong et al., 1992), for example. The mortality rate in the group that had received GLN was 15% (4/26 rats), but the mortality rate in the control group was 46% (13/28). Hong et al. (1992) focused, to some extent, on the role that the preservation of hepatic glutathione (GSH) concentrations are thought to play in the resistance to acetaminophen-induced liver failure, and they discussed some of the research in which GLN availability, in some cell types, had been shown to be rate-limiting, for all practical purposes, for GSH biosynthesis. There is, actually, more of a rationale for the use of GLN as a precursor of GSH in something like acetaminophen-induced liver failure, as noted by the authors, than there is for the use of GLN as a GSH precursor in other contexts. As discussed by Hong et al. (1992), acetaminophen can reduce the overall GSH pool ([GSH] + [GSSG]) and doesn't just disturb the intracellular redox state, as reflected in the [GSSG]/[GSH] ratio. The authors cited research that had shown a [GSSG]/[GSH] ratio of less than .01 in the livers of animals that had been subjected to acetaminophen-induced liver failure, and, even in the presence of that ratio, the absolute levels of reduced GSH (GSH is the reduced state) levels were drastically depleted. But, in many cases, the assumption has been that it's enough to simply replenish the overall GSH+GSSG levels, and that's not necessarily going to be beneficial in many other contexts. But my point was that the hepatoprotective effects were not necessarily a result of the GLN-induced increases in hepatic GSH availability. But I've seen other research that supports the authors' assertion that GLN availability can be limiting for GSH biosynthesis under some conditions. I'd add that there's a lot of research showing that orally or parenterally-administered nucleotides can protect against experimental liver injuries in animals, and I tend to think that nucleotide monophosphates or diphosphates or triphosphates (or triacetyluridine), along with reduced folates, such as L-methylfolate or levoleucovorin, and some amount of methylcobalamin, would be more effective than trimethylglycine (betaine) or phosphatidylcholine or many of the other approaches to liver damage, but that's only my opinion. I've discussed that in past postings. Experimental liver damage is almost used as a kind of generic method, in animals, for evaluating mitochondrial toxicity or any number of other processes, and the research tends to be relevant to many other disease states that involve organs other than the liver.

Those articles, along with other articles, are relevant to an understanding of the way GLN behaves in cells that are largely nonmitotic or postmitotic, such as the brain. GLN is known to be utilized as a precursor of tricarboxylic acid (TCA) cycle intermediates and for other purposes by Kupffer cells and hepatic stellate cells in the liver, especially following a liver injury, and those cells are mitotic (again, especially after an injury). But my point is that it's possible to make crude comparisons between the effects of GLN on the liver and the effects of GLN on the brain. In contrast, the effects of GLN on proliferating lymphocytes can vary throughout the cell cycle, etc.

More specifically, the traditional model of the intercellular compartmentation of glutamine and glutamate metabolism in periportal and perivenous hepatocytes [Souba, 1991: (http://www.ncbi.nlm.nih.gov/pubmed/1892702)] is reminiscent of the traditional model of the glutamine-glutamate cycle in the brain, but neither one of those traditional models is especially helpful in allowing one to understand the effects of exogenous GLN on the brain (or liver). In each case, the traditional model is an oversimplification of the reality, in vivo, and can even become problematic, particularly if one is trying to understand the effects of exogenous (supplemental) GLN in any kind of disease state, as noted by Souba (1991). According to the traditional model, GLN is transported into periportal hepatocytes and deamidated into glutamate by glutaminase, a mitochondrial enzyme that is abundantly-expressed in periportal hepatocytes and that is presented as being expressed to a negligible extent in perivenous hepatocytes (Souba, 1991). Periportal hepatocytes also display more abundant (or, supposedly, exclusive) expression of urea cycle enzymes and utilize glutaminase-derived ammonia in the urea cycle. Perivenous hepatocytes are much less numerous than periportal hepatocytes [Haussinger, 1990: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1131284&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/2185740)] and supposedly are the primary or exclusive subtype of hepatocytes that express glutamine synthetase (Souba, 1991). That type of model would seem to suggest that exogenous GLN would be utilized only or primarily by periportal hepatocytes, but that's unlikely to really be the case. For example, Watford and Smith (1990) [Watford and Smith, 1990: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1131276&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/1970242)] found that perivenous hepatocytes did, in fact, display glutaminase activity, even though the glutaminase activity in periportal hepatocytes was 2.33 times the activity in perivenous hepatocytes. Those authors cited research that had shown that perivenous hepatocytes evidently comprise the only subtype of hepatocytes that expresses GLN synthetase. But the perivenous hepatocytes wouldn't even necessarily have to express glutaminase in order to be influenced by exogenous GLN, however. Many articles have shown that increases in the intracellular GLN concentration, up to 650 uM or so [Smith et al., 1984: (http://www.ncbi.nlm.nih.gov/pubmed/6146632)], at least, are accompanied by decreases in GLN synthetase activity, and Sandrasagra et al. (1988) [Sandrasagra et al., 1988: (http://www.ncbi.nlm.nih.gov/pubmed/2903721)] cited a lot of those articles, including some that have shown an inverse relationship between intracellular GLN concentrations and GLN synthetase activity. But the point is that some of the research has shown that GLN can fairly directly and rapidly, such as within 1.5 hours (Sandrasagra et al., 1988), decrease GLN synthetase activity. The mechanism still isn't known, but it could be that it's an allosteric effect or that the GLN-mediated increase in the proteasomal degradation of GLN synthetase requires the formation of glutaminyl-tRNA, etc. But some of the articles have shown changes in the Vmax in response to GLN, and it's unlikely that the ubiquitination of the GLN synthetase protein, by proteasomal enzymes directed to GLN synthetase by exogenous GLN (via unknown mechanisms), would be responsible for a GLN-induced decrease in the Vmax (Smith et al., 1984) of GLN synthetase, although anything's possible. So exogenous GLN could decrease GLN synthetase activity in perivenous hepatocytes, and that could reduce ATP consumption. Also, 2-oxoglutarate or alanine or aspartate or other substrates, derived from the metabolism of GLN in periportal hepatocytes, could enter perivenous hepatocytes, etc. The point is that the model of the "cycle" in the liver has the potential to be misleading and make it seem as if cells in the liver are limited, in a "strict" way, in their capacities to utilize or respond to exogenous GLN. One can look at research on the GLN-GLT-GABA cycle in the brain and get the same type of misleading sense that GLN can only be used by neurons and that it's going to build up in astrocytes and cause encephalopathy or something. It's ammonia that causes most of those effects, in my opinion, in the context of hepatic encephalopathy. Ammonia causes excessive calcium influx and inhibits multiple mitochondrial enzymes, and the resulting ATP depletion basically cripples the volume regulatory functions of astrocytes and causes astrocytic swelling that is partially a consequence of a cell-energy-metabolism-failure-induced impairment in the export of GLN from astrocytes. But the fundamental problem is not that GLN per se is causing osmoregulatory disturbances. The problem is mitochondrial dysfunction in astrocytes, in my opinion. The beneficial effects of GLN synthetase inhibitors in hepatic encephalopathy could, incidentally, be partly a result of decreases in ATP consumption and not just a result of decreases in GLN formation and in GLN-dependent astrocytic swelling.

Anyway, this posting is getting too long. I also found multiple articles that show that even intravenous GLN infusions tend to not elevate intracellular GLN concentrations in the liver or skeletal muscles, and the articles show the extremely high rate of turnover of GLN. Souba (1991), cited above, cited reference 6, on p. 291, as an example of research that had shown no increase in intracellular GLN in the liver in response to an infusion of GLN under pathological conditions, and that's relevant to the research that supposedly shows no entry of GLN into the brains of humans with traumatic brain injuries (see past postings). It's very likely that GLN did enter the brain in those people. Although Souba (1991) argued that it had been the slow rate of glutamine uptake that had been a limiting factor in the utilization of GLN, that's basically like saying that the rate of increase of intracellular GLN, in response to uptake + synthesis from glutamate or, by transamination, from aspartate, etc., had been less than the rate of decrease, either by glutaminase activity or export or deamidation by that family of non-glutaminase enzymes in the cytosol, etc. That's very similar to the situation at the blood-brain and blood-CSF barriers. The rate of efflux of GLN from the brain is 3-20 times (or something like that) higher than the rate of influx of GLN, but that doesn't mean that no GLN is being transported into the brain (and into astrocytes and neurons) (!). It says absolutely nothing about the amount of GLN that's passing through the interstitial fluid (ISF) in the CNS (to look at the ISF GLN concentration, in view of the research as a whole), especially given the drastic increases in the oxidation of GLN carbons in the TCA cycle, following ischemia, and the countless articles showing no elevations of intracellular GLN or even plasma GLN in response to parenteral or oral GLN. Watford and Smith (1990) discussed the concept that the intercellular cycling of GLN and ammonia in the liver is essentially a futile cycle (or, similarly, in the brain, although the discussions of the GLN-GLT cycle in the brain generally focus on GLT as a substrate of GLN synthetase and only focus on ammonia, as a substrate, in the context of hepatic encephalopathy and other pathological states), although the cycling is clearly not only an ATP-consuming system. But a lot of ATP is consumed. Anyway, the point is that there's a lot of research showing that GLN decreases GLN synthetase activity across small changes in extracellular GLN, and there's also research showing that GLN lessens the glucocorticoid-induced increases in GLN synthetase activity [Hickson et al., 1996: (http://www.ncbi.nlm.nih.gov/pubmed/8945950)]. Another relevant point is that Hickson et al. (1996) cited articles, on p. R1165, in which researchers had found that the glucocorticoid-induced increases in GLN synthetase expression had correlated positively with the degree of muscle atrophy or denervation of the skeletal muscles, in animal experiments. That's important and is another line of indirect evidence that the pathological effects of elevations in GLN synthetase activity are, at least partially, a consequence of increases in ATP consumption, as a result of increases in GLN synthetase activity. But a lot of articles discuss GLN formation as if it's favorable to energy metabolism. In fact, astrocytes oxidize GLN carbons very readily, just as neurons do, and the GLN-GLT cycle can appear to be "robust" or "harmonious" and can actually be consuming large amounts of ATP with very little to show for it, at least in terms of the amelioration of disease states. The articles that have shown the resistance of intracellular and extracellular GLN concentrations to infusions of large amounts of GLN also suggest, in view of the other research that has shown GLN-mediated decreases in GLN synthetase activity, that a lot of the research showing no apparent effect of lower-intensity exercise on GLN metabolism might be sort of missing the point. If one only looks at the plasma GLN level and finds no change, one could erroneously conclude that exercise produced no significant effect on GLN metabolism. One could even do an MRS study and show no change in the intracellular GLN levels, but the point is that there could be a major change in ATP consumption by GLN synthetase, even though lower-intensity, endurance exercise has sometimes been shown to decrease GLN synthetase activity or expression in skeletal muscle myocytes. Hickson et al. (1996) cited some of the other paradoxes. The main point I'd make is that there needs to be more research that looks at the GLN-GLT cycle in the brain in the context of GLN supplementation, given the vast and increasing amounts of clinical research on the effects of GLN supplementation.

Glutamine as an Energy Substrate in the Liver

But I was going to mention that there are also lots of articles showing that glutamine can prevent or ameliorate experimental liver disease and pancreatic exocrine dysfunction in animals, etc. [see this article and the related articles, etc.: (http://www.ncbi.nlm.nih.gov/pubmed/1971031)]. The different effects probably depend on a lot of different variables, such as the dosage and availability of phosphate and uridine, the degree of damage to the mitochondria, the degree of insulin resistance, and other factors. That's part of the reason something like glutamine would probably be more likely to be useful in diffuse brain injuries, such as traumatic brain injuries induced by concussive traumas, or milder ischemia, rather than as an adjunctive approach in severe strokes that are characterized by "raging" cores of necrotic tissue with no functional mitochondria that could oxidize glutamine-derived 2-oxoglutarate, etc. In contrast, purine nucleotides would be expected to be more "durable" in terms of their usefulness in damaged cells, given that purine nucleotides can provide ribose-5-phosphate for the formation of glycolytic intermediates and activate phosphofructokinase in the cytosol and be degraded into uric acid, etc. That presupposes that adenosine receptor activation isn't so deranged as to preclude their usefulness, but a lot of the research on adenosine in strokes doesn't make a distinction between the intracellular and extracellular actions and metabolic fates of adenosine-derived nucleotides. Adenosine that isn't infused too rapidly is not going to just behave like an A1 adenosine receptor agonist, for many reasons that I can't get into. But there are some situations in which there's severe damage or derangement of adenosine receptor signalling, and rapidly-administered, intravenous adenosine can be detrimental under those extreme circumstances. But obviously one would want to discuss these things with one's doctor, even though no one's talking about using intravenous adenosine in a willy-nilly fashion.

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.

Stimulation of Respiration and Activation of TCA Cycle Enzymes by Inorganic Phosphate in Isolated Mitochondria

In this article [Bose et al., 2003: (http://www.jbc.org/cgi/reprint/278/40/39155)(http://www.ncbi.nlm.nih.gov/pubmed/12871940)], Bose et al. (2003) found that inorganic phosphate (Pi) increased the respiratory rate in the isolated mitochondria from pigs' skeletal muscle cells and cardiac muscle cells, and the authors used the increase in the rate of NADH generation as an important indication that the respiratory rate had increased. The increase in NADH generation occurred in the presence of uncouplers (compounds that uncouple the redox reactions of the multienzyme complexes in the electron transport chain with the generation of ATP by the F1F0-ATPase protein) [see here for discussion: (http://hardcorephysiologyfun.blogspot.com/2009/05/cytosolic-redox-potential-and-proton.html)], and Bose et al. (2003) noted that the Pi-induced increases in the rate of NADH generation were likely to have been a result of the "global" activation of various or numerous NAD(+)-dependent dehydrogenase enzymes or enzyme complexes by Pi. The authors cited research, on p. 39161, showing that Pi can activate the NADH-generating TCA cycle enzymes 2-oxoglutarate dehydrogenase, NAD-dependent isocitrate dehydrogenase (this is not the same as the NADP-dependent isocitrate dehydrogenase enzyme). The NADH is then more or less immediately oxidized to NAD+ by respiratory chain enzymes, assuming there's enough oxygen and the mitochondria have not been damaged, etc., and numerous TCA cycle dehydrogenase enzymes either bind complex I, a multienzyme respiratory chain complex that oxidizes NADH formed by TCA cycle enzymes, and channel NADH to complex I or are functionally coupled to complex I activity less directly [Sumegi and Srere, 1984: (http://www.jbc.org/cgi/reprint/259/24/15040.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/6439716)]. Bose et al. (2003) also argued, on p. 39162, that the way in which Pi appears to regulate respiration, by multiple mechanisms, might mean that Pi could exert an antioxidant function
["the generation of free radicals in the mitochondria may be minimized" (Bose et al., 2003, p. 39162], but the authors also cited, on p. 39163 (reference 35), research implying that Pi could exacerbate the augmentation of the rate of free-radical formation following ischemia. I'd wonder what the concentrations used by the authors might have been, in some of those articles cited, because I've seen cell-culture studies showing effects of Pi that don't make sense to me and use supraphysiological concentrations of Pi, show proapoptotic or toxic effects of massive concentrations of Pi, or contrast, in ways that may lack physiological relevance, the effects of excesses of Pi with the supposed protective effects of various drugs, etc. That said, I do think Pi could affect mitochondrial functioning in ways that are not desirable, but it's noteworthy, as Bose et al. (2003) intimated, that ischemia and other forms of metabolic stress can cause Pi to be released during the degradation of phosphocreatine and could derange mitochondrial Pi homeostasis in ways that would be more significant than the ways in which increases in Pi availability would be likely to derange Pi homeostasis. One is unlikely to be able to "hide" from ischemia-induced, wild extremes in mitochondrial Pi influx by restricting dietary Pi, for example, because Pi depletion has the potential to exacerbate those "wild swings" in Pi availability by causing hypoxia, ATP depletion, hemolysis, rhabdomyolysis, etc., in my opinion. But it's worth noting that excesses of intracellular, free Pi could produce adverse effects on mitochondrial functioning.

Bose et al. (2003) also cited research showing that the transport of Pi across the inner mitochondrial membrane is likely to influence the pH gradient across the inner mitochondrial membrane, given that Pi transport appears to be coupled to OH(-) or H(+) transport, and that Pi is used a substrate in the phosphorylation of ADP by the F1F0-ATPase protein. I don't know if there's an enzyme-bound intermediate that's formed from Pi and that contains a hydrolyzable phosphodiester bond, etc. It looks like it wasn't known, as of 2000 [Vinogradov, 2000: (http://jeb.biologists.org/cgi/reprint/203/1/41.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/10600672)]. That's remarkable. That's a terrific article, though, by Vinogradov (2000), and the most important piece of information in there is probably the statement on p. 44 that F1F0-ATPase is "activated" by a free magnesium ion, meaning free Mg(2+), and doesn't just depend on magnesium bound to adenosine nucleotides, as MgATP(2-) and MgADP(-). That could be really important, and it's basically like saying it's a catalytic magnesium ion. That could conceivably help to explain some of the supposed neuroprotective effects of magnesium and could also explain some of its apparent effects on exercise performance, etc. Magnesium also generally enhances overall glycolytic activity and creatine kinase activity, and those effects, along with its purine nucleotide-buffering effects (preventing the loss of adenosine nucleotides, etc.), could also be relevant in those contexts. Never mind that there's an incorrect assumption, in most articles and textbooks, that the intracellular magnesium concentration is high enough to bind to all of the available free ATP and ADP, etc. That's not likely to be true in 99.9 percent of people, and, in my opinion, the activities of many enzymes that contain binding sites for catalytic magnesium ions are likely to be sensitive to changes in magnesium status. Vinogradov (2000) also noted that magnesium is likely to also bind Pi, probably more loosely than magnesium binds some of its other substrates and regulatory factors. I've seen that mentioned in other articles, including the article by Bose et al. (2003), and Bose et al. (2003) cited research, on the first page of their article, describing the capacity of Pi to bind calcium and magnesium (I'm assuming they're talking about reversible binding, in the context of the regulation of intramitochondrial free calcium by its complexation with orthophosphate, etc.) and influence their effects on respiration. I'm not sure what the mechanism is by which Pi activates the TCA cycle enzymes, but I'll have to read on that. Maybe it's partially a result of allosteric effects, and maybe some of those allosteric effects are a result of Pi-induced changes in Ca(2+) binding to the enzymes or enzyme complexes, etc. I'll have to look at some of those articles.

Fractionation of Fixed-Dosage Preparations of Uridine or Triacetyluridine

The context for this posting has to do with the fact that, with some preparations of uridine or triacetyluridine (http://hardcorephysiologyfun.blogspot.com/2009/07/triacetyluridine-uridine-prodrug.html), one is faced with a choice of taking a fixed dosage form intermittently, such as every third day or whatever, taking the whole dosage form daily at a significant cost, or thinking of some way to "fractionate" or parcel out the fixed dosage form of "tang-like" powder. One approach, apart from the freezing of the dosage form dispersed in the liquid, as discussed in that past posting, would be to figure out the approximate "volume," meaning X number of 1/2 teaspoons or X teaspoons or whatever, of the entire dosage form, take so-and-so many 1/2 teaspoons or whatever (so-and-so many "mL" of it), and then just freeze the dry powder in some plastic container. These things might seem strange, but storing it in a plastic bag at room temperature could conceivably allow for bacterial growth, in my opinion, and the condensation formed by the cooling of the air, in a refrigerator, would presumably be greater than that formed in a freezer. I don't know what the best way to do it would be. That way, at least one wouldn't need to worry about intramolecular degradative reactions or oxidative degradation, etc. Anyway, don't shoot the messenger. It's not my fault that it's come down to "tupperware psychopharmacology."

Oculocereborenal Syndrome of Lowe (OCRL): Potential Involvement of Disturbances in Megalin Fnxn's & Relevance to the Effects of Phosphate Depletion

(I meant to type oculocerebrorenal in the title, but my mind isn't working especially well this week, due to the intranasal flu vaccine.) This article [Ramanathan et al., 2009: (http://www.sajaa.co.za/index.php/sajaa/article/viewPDFInterstitial/387/428)] is maybe a poorly-chosen example of an article about Lowe syndrome [the "oculocerebrorenal syndrome of Lowe" (OCRL)], but there are plenty of articles on it (http://scholar.google.com/scholar?q=oculocerebrorenal+Lowe&hl=en). On the surface, the manifestations of OCRL look very similar to Fanconi's syndrome and to the manifestations of intracellular phosphate depletion, but, apparently, many people who have OCRL, caused by hypofunctionality of the OCRL protein(s), or who have a subtype of Dent's disease that is caused by mutations in the OCRL protein (the OCRL protein is an inositol polyphosphate 5-phosphatase that hydrolyzes mainly phosphatidylinositol 4,5-bisphosphate, or PtdIns(4,5)P2, into phosphatidylinositol 4-phosphate but also hydrolyzes other phosphatidylinositols, including phosphatidylinositol 3,4,5-triphosphate, or PtdIns(3,4,5)P3, into phosphatidylinositol 4,5-bisphosphate, etc.) do not display the phosphaturia or hypophosphatemia and rickets that characterize X-linked hypophosphatemic rickets and that occur or can occur in Fanconi's syndrome [Kleta, 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18667737)]. But there's still a lot of overlap with the manifestations of intracellular phosphate depletion and with Fancon's syndrome and other causes of hypophosphatemia. There's hypercalciuria, proteinuria, hypercalciuria, etc., and the proximal tubules are relatively selectively affected, in terms of the effects of the disorder on the kidneys. OCRL also causes vacuolar changes in myelin that fall short of overt demyelination, and OCRL causes cataracts and glaucoma, etc. The encephalopathy and mental retardation in OCRL is seemingly more severe than the effects of hypophosphatemia, but I've cited articles in past postings showing that rather devastating central nervous system damage can result from hypophosphatemia. It's conceivable that intracellular phosphate depletion in neurons and astrocytes, in parts of the brain, is more common than is recognized and that the depletion of phosphate does, in fact, commonly cause vacuolar myelopathy (which, incidentally, is not a very specific neuropathological change and occurs in a variety of contexts, including vitamin B12 depletion, etc.). It's not all *that* generic, as a neuropathological manifestation, however, and OCRL is still similar to Fanconi's syndrome, in my opinion and the opinion of others [Erdmann et al., 2008: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2025683)(http://www.ncbi.nlm.nih.gov/pubmed/17765681)]. It's not really useful to say that it's a totally different condition, when there's so much overlap of the manifestations.

My first thought was that the mutations in OCRL were causing intracellular phosphate depletion with something like intermittent hypophosphatemia, but the most direct manifestation of OCRL (the syndrome) is the aberrant accumulation of PtdIns(4,5)P2, primarily. One possibility is that, in humans who display significant intracellular phosphate depletion and develop proximal tubular acidosis and other changes that overlap with those found in OCRL or Dent's disease caused by mutations in the OCRL protein (Dent's disease can also result from mutations in chloride transporters), there's a generalized depletion of different phosphatidylinositols that, by some mechanism, leads to a similar skewing of the abundances of different phosphatidylinositols in favor of PtdIns(4,5)P2, as in OCRL. Or it's possible that, in OCRL, the impairment in the hydrolysis of PtdIns(4,5)P2 leads to a reduction in the availability of inorganic phosphate for use in ATP formation, much as the sequestration of phosphate in fructose 2,6-bisphosphate can cause ATP depletion following a fructose load. Or, maybe the formation or turnover of mutiple PtdIns's are upregulated in a way that sequesters inorganic phosphate, in multiple pools of PtdIns's, in a way that's maladaptive and detrimental to energy metabolism. Or, it may have nothing to do with energy metabolism.

But Erdmann et al. (2008) found, basically, that the accumulation of PtdIns(4,5)P2 in endocytic vesicles apparently deranged the trafficking of megalin to the apical membranes of proximal tubule epithelial cells. In any case, there can be abnormalities in the uptake of calcium and other constituents of tubular fluid and in receptor-mediated endocytosis by megalin in response to the conditions that occur in people who have OCRL mutations (and the OCRL syndrome) (Erdmann et al., 2008). Erdmann et al. (2008) mention that deranged megalin signalling could account for the CNS abnormalities and that patients with Dent's disease (even the forms due to mutations in chloride transporters) exhibit abnormalities in the functioning of megalin. Erdmann et al. (2008) didn't mention it, but megalin transports vitamin B12 bound to transcobalamin (http://scholar.google.com/scholar?hl=en&q=transcobalamin+megalin) and also transports vitamin D bound to vitamin D binding protein (http://scholar.google.com/scholar?hl=en&q=%22vitamin+D+binding+protein%22+megalin). Megalin serves a transport function across the blood-brain and blood-CSF barriers (http://scholar.google.com/scholar?hl=en&q=megalin+%22blood-brain%22+OR+%22blood-CSF%22), and the "spongy" changes in myelin or "pallor" of myelin (http://scholar.google.com/scholar?hl=en&q=myelin+OCRL+pallor+OR+spongy) seen in people who have OCRL hypofunctionality are reminiscent, in my mind, of the vacuolar myelopathy seen in subacute combined degeneration, due to vitamin B12 depletion (http://scholar.google.com/scholar?hl=en&q=vacuolar+myelopathy+B12), or in humans who have methionine adenosyltransferase deficiency, etc. [(http://scholar.google.com/scholar?hl=en&q=vacuolar+myelopathy+methionine+OR+%22S-adenosylmethionine%22+OR+%22S-adenosyl-L-methionine%22); (http://scholar.google.com/scholar?hl=en&q=myelin+deficiency+%22methionine+adenosyltransferase%22)].

It's interesting that cycloleucine, an inhibitor of methionine adenosyltransferase (MAT), the enzyme that synthesizes S-adenosylmethionine (SAM-e), causes "vacuolation" of myelin (http://scholar.google.com/scholar?hl=en&q=myelin+cycloleucine+vacuolation+OR+vacuolar). It's likely that "vacuolar myelopathy," which can sometimes be characterized by pathological changes in the myelin and also in oligodendrocytes or other cells [such as inclusion bodies in the nuclei of different cell types (http://scholar.google.com/scholar?hl=en&q=%22vacuolar+myelopathy%22+inclusion+body+vacuolation+OR+vacuolar)], is heterogeneous, but one interpretation would be to say that phosphate depletion can reduce SAM-e levels by reducing ATP and adenosine nucleotide pools in oligodendrocytes and other cell types. ATP depletion is known to be capable of causing SAM-e depletion [see either Morrison et al., 1997, or Eto et al., 2002, both of whom showed that SAM-e levels were decreased in the brains of people who had had Alzheimer's disease (the authors in at least one group were saying, correctly, in my view, that the SAM-e depletion was really likely to have been caused by ATP depletion): (http://scholar.google.com/scholar?hl=en&q=ATP+%22severely+decreased%22+Alzheimer%27s+%22S-adenosylmethionine%22)], and that, together with derangements in the abundance of PtdIns(4,5)P2 and other phosphatidylinositols (causing reduced vitamin B12 transport into the brain by reducing the megalin-mediated uptake of B12, etc.), could account for the web of associations I've discussed in this article. It's interesting that Reed et al. (2007) [Reed et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17392004)] found that cats that displayed low serum vitamin B12 and low serum folate levels also tended to display low serum phosphate levels (Reed et al., 2007). One could attribute that to any number of changes and say that the cats had some kind of Fanconi's syndrome that impaired reabsorption of folate binding protein, transcobalamin, and also inorganic phosphate from the tubular fluid. It's known that megalin knockout mice display low-molecular weight proteinuria (http://scholar.google.com/scholar?hl=en&q=megalin+proteinuria), as discussed by Erdmann et al. (2008), and lose different vitamins and other proteins in their urine, and megalin also transports folate binding protein (http://scholar.google.com/scholar?hl=en&q=megalin+folate+binding+protein). But phosphate depletion per se can cause metabolic acidosis or ATP depletion without acidosis in the proximal tubules and could, in my opinion, be a cause and consequence of proximal tubule pathologies. Here's another article that describes an association of B12 depletion with phosphate depletion and that could be explained by the fact that malabsorption, as in liver disease, can cause hypophosphatemia and cobalamin deficiency and also folate depletion [Wojtyczka, 1998: (http://cs.portlandpress.com/cs/095/0735/cs0950735.htm)(http://www.ncbi.nlm.nih.gov/pubmed/9831699)]. Those types of effects, such as loss of vitamin B12 and reduced folates and phosphate in the urine, could explain some of the post-infectious mono issues that people have [(http://scholar.google.com/scholar?hl=en&q=infectious+mono+nephritis+OR+tubular+OR+tubulointerstitial+OR+%22proximal+tubule%22); (http://scholar.google.com/scholar?hl=en&q=infectious+mono+adverse+OR+complication)]. It could be similar to the research showing that cerebral folate deficiency can result from expansion of the pools of antibodies that bind to the reduced folate carrier and other folate transporters at the blood-CSF barrier, given that the immune infiltration of the EBV-infected proximal tubule epithelial cells (http://scholar.google.com/scholar?hl=en&q=infectious+mono+EBV+%22proximal+tubule%22) could create a mess of immune-mediated impairments in proximal tubule functioning (such as by cytokine-mediated disturbances in energy metabolism, etc.). Supposedly, EBV doesn't infect choroid plexus epithelial cells, but I wouldn't be surprised if it did (http://scholar.google.com/scholar?hl=en&q=EBV+%22choroid+plexus%22). There are some significant problems with the notions that a lot of people have about the cell types that EBV supposedly can or can't infect. Here are some more searches [(http://scholar.google.com/scholar?hl=en&q=%22choroid+plexus%22+CD21+OR+C3d+OR+C3R); (http://scholar.google.com/scholar?hl=en&q=EBV+C3R+OR+C3d+OR+CD21)]. Everyone assumes that CD21 isn't likely to be expressed by cells in the CNS and that EBV must infect cells by binding to CD21, but what if it isn't true. A lot of viruses can infect cells using multiple transport mechanisms, some of which have only recently been discovered for influenza, for example. Also, there are significant problems with detecting EBV proteins during autopsies, and many articles look for EBV DNA or viremia (there's not going to be a bunch of viral DNA floating around, all over the place, in a cell latently-infected with EBV). EBV infects epithelial cells in basically every other organ, and it probably infects astrocytes and microglia (http://scholar.google.com/scholar?hl=en&q=EBV+infection+astrocyte+OR+%22human+monocytes%22) and pericytes (http://scholar.google.com/scholar?hl=en&q=resident+macrophage+pericyte+brain) and fibroblasts (i.e. meningeal fibroblasts, probably) [see Koide et al., 1997: (http://scholar.google.com/scholar?hl=en&q=EBV+fibroblasts)]. That type of effect on the proximal tubules could reduce phosphate and vitamin D and reduced folate and vitamin B12 reabsorption by the kidneys and could produce similar impairments at the blood-CSF barrier, etc. (http://scholar.google.com/scholar?hl=en&q=infectious+mono+brain+complication+OR+adverse).

I don't claim to be able to explain all of the different manifestations of these conditions, but the overlap of the effects of OCRL mutations with the effects of idiopathic Fanconi's syndrome and also the effects of intracellular phosphate depletion are fairly difficult to ignore completely. There must be some explanation, but it's interesting, in any case.

Precipitation of Free Zinc by Phosphate in Ex Vivo Brain Tissue: Potential Relevance to Zinc-Induced Neurotoxicity

This article [Rumschik et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19183267)] is interesting, and Rumschik et al. (2009) found that inorganic phosphate (Pi) can chelate zinc and reduce the influx of free zinc into neurons in ex vivo preparations of brain tissue. This is relevant to Alzheimer's disease and other neurodegenerative conditions (and also to depression and other psychiatric conditions, etc.), in my opinion, because an excess of free zinc is generally very toxic to cells and can shut down oxidative metabolism and induce necrotic or apoptotic cell death (see past postings). Rumschik et al. (2009) also found that histidine can enhance the "solubility" of zinc, in the sense that the complex formed by the chelation of zinc by histidine can be transported into cells through dipeptide or amino acid transporters. That's not exactly an enhancement of the solubility of zinc but just means that the zinc-histidine (zinc histidylate or whatever) complex is more soluble than the zinc-phosphate complex(es) are. Glutamine did not meaningfully enhance the solubility, and hence the influx, of zinc, and that's a good thing. The authors mentioned that insoluble, extracellular zinc-phosphate complexes could conceivably become pathological and serve as a site for the nucleation or seeding of Abeta-peptide-containing, extracellular plaques (i.e. a pro-aggregating effect), etc., but I would guess that maintaining an adequate amount of intracellular phosphate in neurons and in the CSF would, in comparison to the effects of intracellular (and extracellular, to some extent) phosphate depletion, tend to exert a net neuroprotective effect. That's just my opinion, but the research has generally shown that intracellular Pi depletion produces neuropathy and neuropathological effects by multiple mechanisms. I tend to think Pi could exert similar effects intracellularly, especially given that the intracellular Pi levels are higher than the extracellular Pi levels. The authors mentioned that the assumption, in the context of ex vivo or in vitro research, has been that the extracellular and CSF Pi levels are around 1 mM, but the human CSF Pi concentration is apparently around 0.47 to 0.50 mM, under normal circumstances. That's potentially important, because, even though the steady-state intracellular and extracellular fluid Pi levels, in different cell types, have generally been found to be relatively independent of one another, the intracellular Pi concentration can correlate with and increase in response to boluses (even small "boluses," meaning significant, single dosages). A relatively higher extracellular fluid Pi concentration might more effectively buffer the intracellular Pi levels during miniature "Pi crises," such as after exercise or, more significantly, after ischemic insults (exercise produces mild ischemia in many organs, but I'm referring to would-be brain injuries, here). There's a tendency to think that all values within a normal range of values, for a blood test or physiological parameter, are equally "good" or equally "normal," but the research on the extracellular and intracellular levels of uric acid and other compounds has shown that this can be a problematic assumption. Small changes in the extracellular uric acid levels can drastically affect the rates of nitric oxide output (and, hence, fairly directly, the peroxynitrite output) by the nitric oxide synthases and the macrophages or monocytes that express those enzymes.

I should mention that increases in acidity, meaning an increase in H(+) availability, can enhance the release of zinc from storage vesicles [Colvin et al., 2000: (http://crab-lab.zool.ohiou.edu/colvin/neurochem.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/10762091); Colvin, 2002: (http://ajpcell.physiology.org/cgi/content/full/282/2/C317)(http://www.ncbi.nlm.nih.gov/pubmed/11788343)], and one could make the argument that the potential for an alkalinizing effect of Pi supplementation, such as in the lower dosage range, on the extracellular fluid could further reduce the kinds of wild fluctuations in free zinc concentrations that can be neurotoxic. But the pH dependences of zinc influx and zinc release are complex (Colvin, 2002), and that suggestion of mine is likely to be an oversimplification. Nonetheless, Pi obviously plays a general role in buffering pH changes, and that acid-base buffering effect could be protective against zinc-mediated neurotoxicity.

Effects of Changes in Phosphate Availability on Glutamatergic Transmission: Potential Relevance to Alzheimer's or Age-Associated Cognitive Impairment

This article [Glinn et al., 1997: (http://www.ncbi.nlm.nih.gov/pubmed/9200502)] is really interesting, and the authors found that the ATP levels in cultured neurons correlated positively, up to a point, with the availability of inorganic phosphate (Pi). The concentration-dependences found by the authors for that and other correlations, such as of metabolite concentrations with Pi availability, seem to have the potential to be misleading, because many articles have shown that the intracellular ATP or 2,3-bisphosphoglycerate levels do increase in response to seemingly-insignificant, acute increases in extracellular Pi availability (see past postings). It's probably that the availabilities of energy substrates in the culure medium provide the cells with everything they need, and those conditions are unlikely to prevail in vivo. Also, there's some strange issue with the concentrations of free Pi being found by different groups of researchers. Glinn et al. (1997) and other groups have found concentrations in the 30+ mM range, but others have found that the free Pi levels are between 0.8 and 4 or so mM. Maybe there are differences between cell types, but those seem like awfully large differences. I would think it would be difficult for anyone to determine the percentage that would exist unbound, at any given concentration. I'll have to read up on that.

Glinn et al. (1997) also mention some really interesting research suggesting that low Pi availability to the brain may contribute to cognitive dysfunction in people who go on to develop Alzheimer's (reference 30, cited on page 91). They also discuss research showing that Pi availability may help protect against glutamatergic neurotoxicity (i.e. "excitotoxicity"). They mention, earlier in the article, that Pi can be utilized by 3-phosphoglycerate kinase and pyruvate kinase, and I looked into that topic a little bit. In that context, it's interesting that the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK) form a (dimeric?) enzyme complex, and the 1,3-bisphosphoglycerate (1,3-BPG) that is formed from glyceraldehyde-3-phosphate (GAP), by GAPDH, is then channeled, apparently, to PGK and converted into 3-phosphoglycerate [Ikemoto et al., 2003: (http://www.jbc.org/cgi/content/full/278/8/5929)(http://www.ncbi.nlm.nih.gov/pubmed/12488440?dopt=Abstract)]. What's interesting is that, essentially, Pi is used to fairly directly (via its incorporation into 1,3-BPG) phosphorylate ADP into ATP, and the ATP formed by the GAPDH-PGK complex is preferentially used to transport glutamate into presynaptic vesicles (exogenous ATP is not as effective in promoting vesicular glutamate transport) (Ikemoto et al., 2003). A lot of researchers refer to that ATP-requiring transport as "glutamate uptake," but that terminology could potentially cause one to confuse the process with synaptic glutamate uptake. Ikemoto et al. (2003) are talking about the vesicular glutamate transport that "loads" glutamate in presynaptic vesicles for release. There's other research showing that mitochondrial glutaminase (and the mitochondria that contain it) is localized at the sites of synaptic glutamate uptake and that Pi availability, especially insofar as its availability is important for the activation of glutaminase in astrocytes, plays a role in maintaining synaptic glutamate uptake. Some of those articles that cite Glinn et al. (1997) look interesting (http://scholar.google.com/scholar?cites=5143060761810849298&hl=en).

Phosphate Depletion Associated With Hypoxia, Autonomic Neuropathy, Hypoventilation, or Paralysis: Potential Relevance to Sudden Infant Death Syndrome

These [Siddiqui and Bertorini, 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9572247); Gravelyn et al., 1988: (http://deepblue.lib.umich.edu/bitstream/2027.42/27325/1/0000348.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/3364446); Steckman et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16642427); Heames and Cope, 2006: (http://www.ncbi.nlm.nih.gov/pubmed/17090245)] are some interesting articles that show some of the variegated manifestations of hypophosphatemia. A crucial fact that I've taken from the research on phosphate homeostasis (it's arguably the most crucial point) is that neither the steady-state nor the between-dosage (in the context of phosphate infusions in animals or phosphate supplementation in humans) intracellular phosphate levels, in either muscle cells or red blood cells, correlates with the serum phosphate levels. For example, Chobanian et al. (1995) [Chobanian et al., 1995: (http://www.ncbi.nlm.nih.gov/pubmed/7900836)] found that the intracellular ATP concentrations in cells in the proximal tubules correlated positively with the intracellular inorganic phosphate (Pi) concentrations, and artificially-induced changes in extracellular Pi concentrations produced changes in the intracellular Pi concentrations. But in human studies, the intracellular Pi values generally do not correlate with serum Pi values, and the intracellular Pi concentrations can be significantly depleted in a person who has a normal serum Pi level.

That usual absence of a correlation between intracellular and serum Pi concentrations means, in my opinion, that intracellular phosphate depletion, in "normophosphatemic" people, should be considered as a possible factor contributing to some of these conditions that have been associated with hypophosphatemia. Siddiqui and Bertorini (1998) cited research showing that phosphate depletion can produce neuropathy that mimics Guillain-Barre syndrome, and the authors described the symptoms of a patient who developed neurological symptoms after she had been given parenteral nutrition without phosphate. The manifestations of neuropathy were suggestive of demyelinating polyneuropathy but were rapidly reversed by phosphate supplementation, meaning that there wasn't demyelination. The authors also discussed the fact that an increase in hexokinase activity, in response to insulin that has been released after the intake of carbohydrates, is thought to be an important factor that mediates the carbohydrate-induced increase in the transport of phosphate into cells and the decrease in serum phosphate that can result from that transport (Siddiqui and Bertorini, 1998). The authors also cited research showing cognitive dysfunction and encephalopathy in hypophosphatemic or (merely) intracellular-phosphate-depleted people (Siddiqui and Bertorini, 1998). One interpretation of the article by Steckman et al. (2006), in which gallstone-induced pancreatitis occurred in conjunction with hypophosphatemia and improved in response to phosphate administration, is that the phosphate depletion was causing neuropathy and interfering with gallbladder contractions. Neuropathy is known to be associated with gallbladder disease, and the normal functioning and contraction of the gallbladder is regulated by its autonomic (and sensory) innervation [(http://scholar.google.com/scholar?q=neuropathy+gallbladder+gallstone&hl=en);
the visceral sensory innervation can influence mast cell degranulation in the gallbladder, via the efferent-action-potential-mediated release of neuropeptides, and changes in mast cell degranulation and neuropeptide release can influence the autonomic regulation of gallbladder functioning, etc.: (http://scholar.google.com/scholar?hl=en&q=%22mast+cell%22+gallbladder+CGRP+OR+%22substance+P%22+OR+%22vasoactive+intestinal+peptide%22)]. Another interpretation would be to say that the phosphate depletion caused ATP depletion in the liver and led to cholestasis, etc. Similarly, the respiratory muscle weakness found in association with hypophosphatemia or low serum phosphate levels (Gravelyn et al., 1988, cited above) could be a result of autonomic dysfunction, particularly given that hypophosphatemia can cause reversible quadriparesis (paralysis, meaning the people are transiently quadripalegics) (http://scholar.google.com/scholar?hl=en&q=quadriparesis+hypophosphatemia). The hypoventilation that can accompany hypophosphatemia could also be due to autonomic neuropathy and ATP depletion in parts of the brain (http://scholar.google.com/scholar?hl=en&q=hypoventilation+hypophosphatemia). Hypophosphatemia has also shown up in association with extrapontine myelinolysis (which is central "pontine" myelinolysis that doesn't occur in the pons, essentially), one of the forms of osmotic demyelination that can result from the excessively-rapid correction of hyponatremia with intravenous, hypertonic saline [Qadir et al., 2005: (http://www.jpma.org.pk//PdfDownload/759.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/16045098)]. The authors suggested that ATP depletion in glial cells in parts of the brain might have contributed to the case, but I'm not sure that the authors actually said that the phosphate might have contributed to or caused the ATP depletion. The intracellular phosphate may well have been depleted in parts of the brain, and that depletion may have impaired volume regulation and predisposed to the osmotic demyelination.

In any case, I found this article showing "sinusoidal" seasonal changes in the incidence of sudden infant death syndrome (SIDS) (the seasonal change in the incidence shows up in the Southern and Northern hemispheres, and SIDS was found to peak in the winter in both hemispheres) [Douglas et al., 1996: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2351134)(http://www.ncbi.nlm.nih.gov/pubmed/8646093)], and there's old research suggesting an association of SIDS with vitamin D depletion or differences in vitamin D metabolism or rickets, etc. [Schluter, 1996: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2352183&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/8842097); (http://scholar.google.com/scholar?hl=en&q=%22sudden+infant+death%22+%22vitamin+D%22)] (or with other light-associated changes, such as involving changes in melatonin levels induced by sleeping on the back as opposed to the side, etc.) (Douglas et al., 1996). There's also research showing that infants who were experiencing apnea were more likely to be hypercalcemic than infants not experiencing apnea [Kooh and Binet, 1990: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1452283&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/2207905)]. I couldn't get results to show up on a quick search, but hypercalcemia has been found to occur in hypophosphatemic people. Although Kooh and Binet (1990) didn't find that serum phosphate levels were associated with apnea in any way, the serum Pi levels wouldn't have to. Given that intracellular Pi levels do not reliably correlate with serum Pi levels and that phosphate depletion is known to be capable of causing respiratory paralysis/hypoventilation and hypoxia and neuropathy [see this and many others, some of which I discussed above: Weber et al., 2000: (http://www.ncbi.nlm.nih.gov/pubmed/10663486)] [and given that vitamin D depletion is known to be a cause of phosphate depletion (and that vitamin D supplementation, even in the absence of any genetic defect specifically involving vitamin D receptor signalling)], one possibility is that intracellular phosphate depletion in parts of the brain (and in the red blood cells, causing low-level hypoxia that might gradually have more severe consequences) could contribute to some cases of SIDS. Although there was one small study showing no apparent depletion of 25-hydroxyvitamin D levels in the context of SIDS, there could very easily be different degrees of intracellular phosphate depletion among infants with the same 25(OH)D levels. And looking at the serum phosphate levels wouldn't necessarily show anything, given the lack of correlation of intracellular and serum Pi levels. Someone would have to use MRS scans or look at the intracellular 2,3-DPG or Pi levels in red blood cells in infants, instead of just looking at the serum Pi. It's interesting that Heames and Cope (2006) (cited above) found that they could reduce the rate of infusion of noradrenaline in a manner that was proportional to the increase in serum phosphate, in a person who had developed transient heart failure from postsurgical phosphate depletion. The phosphate depletion basically caused hypotension, and the interactions with noradrenaline are really interesting (the usual thing people discuss is the fact that adrenergic drugs decrease serum phosphate by promoting phosphate uptake into cells). Given the changes in the autonomic regulation of blood pressure that occur in response to changes in the orientation of the body, such as in a baby sleeping prone vs. supine (http://scholar.google.com/scholar?q=autonomic+orthostatic+prone+supine&hl=en), it's possible that there's a kind of feed-forward depletion of intracellular phosphate in parts of the brain that can lead to apnea and then increased ventilation to compensate (and then phosphate depletion because of that and because of the noradrenaline released in response to that, as in the stress response to hypoxia, and to the potential vitamin D-depletion-induced renal phosphate wasting, etc.).

Arguably, the most well-established cause of hypophosphatemia is alkalosis induced by hyperventilation (http://scholar.google.com/scholar?q=hyperventilation+alkalosis+hypophosphatemia&hl=en), and apnea commonly occurs in response to post-hyperventilation alkalosis (http://scholar.google.com/scholar?q=hyperventilation+apnea&hl=en). So the alkalosis, in response to hyperventilation (as in response to autonomic dysfunction during sleep, resulting from changes in the sleep position and from phosphate depletion in neurons or smooth muscle cells or muscle cells in the diaphragm), could drive phosphate into cells outside the brain, thereby reducing phosphate availability to the brain, and then that could gradually set the stage for more severe episodes of hypoxia, more autonomic dysfunction due to the phosphate depletion in the brain, etc. There's evidence of repeated episodes of hypoxia in some research on SIDS [see Takashima et al. (1978) and Rognum et al. (1991): (http://scholar.google.com/scholar?hl=en&q=%22sudden+infant+death%22+hypoxia)]. A decrease in the responsiveness of smooth muscle cells (or other cell types, as in neurons in the brainstem, in the context of phosphate depletion) to noradrenaline occurs in people who have orthostatic hypotension and other derangements of baroreceptor functioning, and L-threo-3,4-dihydroxyphenylserine (DOPS) has been researched as a treatment for orthostatic hypotension and orthostatic tachycardia (DOPS is a precursor of noradrenaline) (http://scholar.google.com/scholar?hl=en&q=orthostatic+DOPS). Hypophosphatemia has been associated with instability in blood pressure, in association with postural hypotension and other problems with the sensitivity and functioning of the baroreceptor reflexes (http://scholar.google.com/scholar?hl=en&q=orthostatic+hypophosphatemia). Anyway, I just put those types of crude thoughts up on this blog.

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."

More Information on Phosphate Homeostasis

In this letter [Roestel et al., 2004: (http://ajp.psychiatryonline.org/cgi/content/full/161/8/1499-a)(http://www.ncbi.nlm.nih.gov/pubmed/15285984)], Roestel et al. (2004) discuss research showing that serum phosphate correlates negatively with symptoms of anxiety and with physical complaints, etc. Roestel et al. (2004) cite this article [Maddock et al., 1987: (http://www.ncbi.nlm.nih.gov/pubmed/3659218)] as evidence of that, and there's a considerable amount of research showing that hypophosphatemia commonly is associated with various psychiatric disorders (http://scholar.google.com/scholar?hl=en&q=hypophosphatemia+psychiatric+OR+psychiatry), especially panic disorder. Some of those articles discuss the capacity of an elevated rate of lactate formation, from various tissues, to contribute to phosphate depletion, and that's been discussed in the context of exercise-induced phosphate depletion (http://scholar.google.com/scholar?hl=en&q=hypophosphatemia+lactate+exercise). Adrenaline, by its activation of beta-adrenoreceptors, in particular, is also thought to contribute to phosphate depletion in the long term and to short-term decreases in serum phosphate, given that adrenergic stimulation tends to increase the uptake of phosphate into cells (i.e. skeletal muscle cells, etc.). The same effect could result from chronic psychological stress, given that even anticipated stress can increase the firing rates of noradrenergic neurons in the locus ceruleus, for example. The authors of a lot of articles discuss the fact that hypophosphatemia tends to be associated with irritability, even in people who do not have panic attacks or whatever other symptoms (http://scholar.google.com/scholar?q=hypophosphatemia+irritable+OR+irritability&hl=en). As vague as that may sound, a lot of these articles discuss those types of excessively excitatory states in the context of hypophosphatemia or relative phosphate depletion. It's interesting that Nanji et al. (1985) [Nanji et al., 1985: (http://www.ncbi.nlm.nih.gov/pubmed/4045178)] suggested that phosphate depletion may have been a cause and not (just) a consequence of liver failure/liver-cell necrosis in one case, and Quirós-Tejeira et al. (2005) [Quirós-Tejeira et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16101727)] found that the normalization of serum phosphate levels paralleled the normalization of liver function in children who had experienced liver damage. Fructose is known to induce ATP depletion by sequestering phosphate, for example, and the point is that hypophosphatemia may actually be, in part, a cause and not just a consequence of some of these conditions that have been associated with hypophosphatemia.

I tend to think that something like ATP disodium might be safer than sodium phosphate for correcting phosphate depletion (in part because some it is likely to be absorbed intact, by paracellular diffusion, and because oral ATP has been shown to elevate adenosine diphosphate and monophosphate levels in the portal venous blood in animals, implying that less of the ATP-derived phosphate might remain in the GI tract and bind calcium and magnesium; there are several other reasons I say that), but it's important to discuss this with one's doctor. Phosphate can inhibit the absorption of calcium, magnesium, iron, and probably other minerals and could, particularly but not exclusively at higher doses, cause life-threatening hypocalcemia in people whose mineral metabolism is already deranged, etc. The kidneys and GI tract and calciotropic hormone system also tend to adapt rapidly to phosphate supplementation, and it seems like it would be more effective, in the long term, in my opinion, to consider some kind of intermittent "challenge" with something like ATP, in combination with resistance exercise, than to try to use sodium phosphate. I actually don't know much about phosphate supplementation, though, and I've never used sodium phosphate and don't intend to. Bremner et al. (2002) [Bremner et al., 2002: (http://www.ncbi.nlm.nih.gov/pubmed/12135811)] found that 4 grams per day of sodium phosphate (Na2HPO4) increased serum phosphate by 30 percent and caused a 25 percent increase in the (intracellular) 2,3-bisphosphoglycerate (BPG) levels in red blood cells (RBC's). BPG alters the affinity of hemoglobin for oxygen and improves the "unloading" of oxygen from hemoglobin, and that's a fairly significant effect. Many other articles have shown similar effects, and Bremner et al. (2002) noted that the steady-state increase in RBC BPG appears to require two or three days to occur at that dosage range (3-4 grams per day). Bremner et al. (2002) also found that the RBC BPG levels didn't correlate with the serum inorganic phosphate concentrations, and that absence of a correlation between serum phosphate and intracellular phosphate levels has been found by many other researchers for many other cell types, such as skeletal muscles, etc. I've discussed that in past postings, and I've also discussed the fact that calcium supplementation tends to bind phosphate in the GI tract and could thereby cause phosphate depletion. Heaney and Nordin (2002) [Heaney and Nordin, 2002: (http://www.ncbi.nlm.nih.gov/pubmed/12074251)] discuss that, and it's interesting that a lot of the assumptions about phosphate metabolism and phosphate intake have ended up being much more complicated than people had previously thought they would be. Hypophosphatemia can cause lactic acidosis, for example, but the correction of the acidosis with bicarbonate has the same effect as BPG depletion from RBC's has on oxygen unloading from hemoglobin and can compound the existing BPG depletion in people who have hypophosphatemia, with disastrous consequences [Jacob, 1975: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1129800&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/1136448)]. But phosphate supplementation can gradually elevate serum bicarbonate and cause compensated metabolic alkalosis. Those types of interactions with acid-base regulation could account for some of the cases in which hypophosphatemia has been associated with idiopathic intracranial hypertension (or, rather, not-idiopathic) (http://scholar.google.com/scholar?hl=en&q=hypophosphatemia+papilledema+OR+%22intracranial+pressure%22+OR+pseudotumor+OR+pseudotumour+OR+%22intracranial+hypertension%22). The rate of CSF formation by the choroid plexuses can be dysregulated by acid-base dysequilibria across the blood-CSF barrier and has been treated by acetazolamide and other carbonic anhydrase inhibitors [see Horovitz et al., 1985: (http://scholar.google.com/scholar?hl=en&q=acid+base+dysequilibrium+papilledema+OR+%22intracranial+pressure%22+OR+pseudotumor+OR+pseudotumor+OR+%22intracranial+hypertension%22)]. It's conceivable that some of the psychiatric symptoms associated with hypophosphatemia are actually, in part, a result of mild elevations [as discussed in this posting, for example: (http://hardcorephysiologyfun.blogspot.com/2009/07/dimming-of-vision-in-depression.html)] in intracranial pressure. There are other paradoxical aspects of phosphate homeostasis, such as the fact that alkaline phosphatase activity is pH-sensitive, to a potentially-important degree, and is inhibited by inorganic phosphate. So phosphate derived from the hydrolysis of pyrophosphate, by smooth-muscle cell alkaline phosphatase, would tend to increase the risk of calcification, by locally diminishing the inhibitory effect of pyrophosphate, but phosphate depletion could conceivably augment alkaline phosphatase activity by causing metabolic acidosis and excessive phosphate turnover, because of adenosine nucleotide deamination in the context of wild swings, up and down, in intracellular free inorganic phosphate concentrations? I don't know, but it's complicated. And the relationship between serum phosphate or extracellular fluid phosphate and calcification risk is "U-shaped," as various authors have noted. In any case, a lot of the research is interesting.

Vitamin D and Phosphate: Relevance to Muscle Weakness in Vitamin D Depletion & Significance of Lipoprotein Binding of Orally-Administered Vitamin D

Chudley et al. (1981) [Chudley et al., 1981: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1862641&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/6793223)] noted that muscle weakness and low-level indications of peripheral neuropathy (and overt neuropathy, but the authors don't really discuss that) can occur in people who are hypophosphatemic, and it's interesting that researchers have commonly found muscle weakness and balance problems (predisposing to falls in elderly people) and muscle pain to be associated with vitamin D deficiency (given that vitamin D supplementation is used to treat hypophosphatemia due to a number of different causes). There's actually evidence that the phosphate depletion that results (in part) from the secondary hyperparathyroidism that, in turn, results from vitamin D deficiency does contribute to muscle weakness and the associated problems (muscle pain, balance problems) (http://scholar.google.com/scholar?q=phosphate+%22vitamin+D%22+muscle+weakness&hl=en). It actually seems likely that the muscle weakness could partly be due to neuropathy induced, in part, to the phosphate depletion that accompanies vitamin D deficiency. I should mention that I don't think vitamin D (or rather the elevations in 25-hydroxyvitamin D) produced in response to UVB is as calcemic as oral vitamin D is, and I base that statement on various lines of evidence. I think it might be that oral vitamin D is converted into 25-hydroxyvitamin D and then into 1alpha,25-dihydroxyvitamin D locally, in enterocytes in the GI tract (the cytochrome P450 enzyme(s) that display(s) vitamin D 25-hydroxylase activity is/are expressed in a wide variety of tissues), or it may be a result of the fact that the oral vitamin D becomes more highly bound to lipoproteins [Haddad et al., 1993: (http://www.pubmedcentral.nih.gov/picrender.fcgi?pmid=8390483&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/8390483)]. That could cause its transport into cells to be regulated in different ways, etc. That article by Haddad et al. (1993) is great, and they say that vitamin D3 produced in the skin (and not just 25-hydroxyvitamin D produced in the liver) is primarily bound to vitamin D binding protein (Gc globulin), but orally-administered vitamin D becomes bound to chylomicrons and lipoproteins and enters the liver much more rapidly than skin-derived vitamin D. The issue wouldn't just be the rate of transport into the liver, though, because the lipoprotein-bound vitamin D and 25-hydroxyvitamin D would enter cells in ways that would not be subject to the same endosomal regulatory mechanisms, etc., as the transport of vitamin-D-binding-protein-bound vitamin D or 25-hydroxyvitamin D would be subject to.

Regardless of the mechanisms, there does seems to be some difference that, in my opinion, makes oral vitamin D more calcemic, and that could be important in the context of some of these issues related to the effects of vitamin D repletion on phosphate homeostasis. I remember reading an old article in which someone suggested that some of the neuroprotective effects of vitamin D, in the context of in vitro or animal experiments, might be mediated by its effects on phosphate homeostasis (on the preservation of ATP, as a result of its phosphate-"sparing" effects and effects on phosphate transport, etc.), but I forget what the article was specifically testing (and I can't find it right now). There would be a limit to the supposed beneficial effects of vitamin D repletion on phosphate homeostasis, and the concomitant elevations in serum calcium could, to some extent, negate the benefits associated with the vitamin D-mediated reductions in urinary phosphate loss (an effect that is, in part, secondary to the localized conversion of 25-hydroxyvitamin D into hormonal vitamin D, in the parathyroid tissue, and autocrine/paracrine suppression of parathyroid hormone release, etc.).