Glutamine and Uridine in Hexosamine Biosynthesis and Glycosylation Reactions

There are lots of interesting articles that have shown the roles that glutamine (GLN)-mediated increases in the O-glycosylation, with beta-N-acetylglucosamine, of serine and threonine residues on proteins can play in mediating either the protective effects or undesirable effects of GLN (http://scholar.google.com/scholar?q=glutamine+hexosamine+ischemia&hl=en). GLN is a substrate of glutamine: fructose-6-phosphate amidotransferase (GFAT), and that enzyme forms glucosamine-6-phosphate and glutamate [Broschat et al., 2002: (http://www.jbc.org/content/277/17/14764.full)(http://www.ncbi.nlm.nih.gov/pubmed/11842094?dopt=Abstract)]. In any case, the overall point is that some of the GLN-mediated protection against damage due to ischemia have been shown to be a result of the augmentation of hexosamine formation by GLN (see that first search), but I've also seen articles showing that GLN can sometimes worsen the course of experimental fatty liver disease or the courses of other disease states, in animals, in which insulin resistance features prominently. There's a large amount of research showing that glucosamine can cause insulin resistance in animals (http://scholar.google.com/scholar?hl=en&q=glucosamine+liver+OR+insulin+OR+ATP) and other undesirable effects, but, under normal circumstances, I remember reading that only about 3 percent of the intracellular GLN in cells in the liver is metabolized into glucosamine. As I've discussed in past postings, the formation of uridine diphosphohexosamines can sometimes sequester large amounts of uridine in ways that is undesirable, in animal models of liver disease, and GLN can also serve as a substrate for de novo uridine biosynthesis. That's not generally something that one wants to accelerate in an unregulated way. But my point would be that, at reasonable dosages, the formation of glucosamine or carbamoyl phosphate, as a precursor of orotate and uridine, from GLN would be processes that would be subject to substantially more regulation than the formation of hexosamines and orotate from exogenous glucosamine and...orotate would be.

Another important point is that it's necessary to take into account the potential for GLN-mediated decreases in glutamine synthetase (GS) activity, with regard to the supposed ATP-sparing effects of that suppression, to occur and to consider the effects of GLN-derived 2-oxoglutarate on mitochondrial ATP formation. One can say that the effects of GLN are mediated by glycosylation during ischemia, but how was the uridine pool preserved during ischemia? The GLN-mediated preservation of ATP could indirectly preserve the UDP-N-acetylglucosamine and overall UDP-hexosamine pools during ischemia, given that ATP depletion tends to lead to loss of pyrimidine nucleosides, either by export or degradation. That's only one example. Not surprisingly, there's actually some research showing that some of the protective effects of uridine in cultured astrocytes, or something like that, are mediated by glycosylation of various proteins, and I remember downloading a paper that shows that glycosaminoglycan formation is more sensitive to increases in uridine availability than other UDP-sugar-dependent or UDP-hexosamine-dependent glycosylation reactions are. Again, however, one has to consider the increases in glucose uptake that exogenous uridine can produce. Did the uridine-induced increases in protein glycosylation exert protective effects by increasing the glucose uptake, or did the uridine-induced increases in glycogen formation, in the face of increase in glucose uptake by other mechanisms, buffer ATP levels and thereby maintain the normal, relative amounts of different UDP-hexosamines that are required for glycosylation reactions that produce other protective effects? Similarly, one can't look at an article on GFAT overexpression in mice, see a lot of adverse effects, and conclude that GLN is going to produce the same effects as GFAT overexpression will (for the reasons I discussed above, involving energy metabolism, primarily). But another important point is that, in some of those articles using high doses of GLN, one has to consider the cumulative, potentially-depleting effects of GLN-induced hexosamine and UDP-hexosamine formation on the intracellular and even plasma inorganic phosphate pools. I have at least one article showing that exogenous uridine can deplete the inorganic phosphate pool, and I'll try to put it up. Another thing to consider would be the use of uridine, glutamine, and inorganic phosphate in some sort of combination approach. The uridine could suppress de novo pyrimidine biosynthesis and avoid some of the undesirable effects of a high rate of de novo pyrimidine (uridine) formation (as discussed in past postings, orotate has tended to lead to ATP depletion in animal experiments) and also prevent the sequestration of uridine, some of which is obviously required for glycogen formation, in UDP-hexosamines. But the goal, in my opinion, should really be to normalize the availability of GLN to the brain or skeletal muscles, in order to prevent unnecessary exercise-induced ATP depletion, etc. There can be a significant increase in ATP turnover in the brain and, obviously, skeletal muscles during high-intensity exercise. That's separate, to a large extent, from the issue of ischemia.

Rambler on Magnesium and Sad Ironies

In this article [Vink et al., 1988: (http://www.jbc.org/cgi/reprint/263/2/757.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/3335524)] found that the depletion of intracellular free magnesium (Mg2+) correlated positively with the magnitude of the damage that was produced by experimental brain injuries in rats, and the administration of intravenous Mg2+, 5 minutes before the injuries, prevented much of the damage. The authors cited some interesting data on the pH dependence of the calculations, based on 31P-MRS data, of the intracellular free Mg2+ values, and the authors used data on the dissociation constant of MgATP(2-) at pH 7.2 (50 uM). It's interesting that the mean pre-injury, intracellular free Mg2+ concentration was 1.01 mM (1,010 uM), and the mean concentration was 0.26 mM (260 uM) by 3 hours post-injury. Vink et al. (1988) also cited research (reference 13, cited on p. 761) that had shown the rate of DNA synthesis in cultured fibroblasts to decrease logarithmically at intracellular free Mg2+ concentrations below 0.24 mM (240 uM). In that article, the rate of protein synthesis was down to almost nothing at those low concentrations, also. Resnick et al. (1997) [Resnick et al., 1997: (http://hyper.ahajournals.org/cgi/content/full/30/3/654)(http://www.ncbi.nlm.nih.gov/pubmed/9322999?dopt=Abstract)] found that the intracellular free Mg2+ levels were inversely correlated with the ages of people, and that means the levels decrease as people get older. It's interesting that the mean concentration of intracellular free Mg2+ in people who were hypertensive was 0.284 mM (284 uM), and the mean concentration in normotensive controls was only 0.383 mM (383 uM). One could argue that the rate of DNA synthesis in mitotic cells (fibroblasts) is going to be much higher at specific points in the cell cycle, but then why are the intracellular free Mg2+ levels in the brains of normal rats 3-4 times the levels in the brains of humans? I'll bet one reason is that laboratory animals generally receive higher intakes of magnesium, in addition to phosphate, etc. One could make the argument that the higher zinc or copper intakes of animals eating "rat chow," or whatever, would cause some neurotoxicity and balance out the benefits that have sometimes been associated with higher Mg2+ intakes. But the discrepancies between rat and human diets tend to not be as significant for some of those metals, like copper and zinc. That doesn't sound like a very good situation, with intracellular free Mg2+ concentrations being that low. This basic search on magnesium in relation to neuroprotection or neurotoxicity yielded 45,000+ results (http://scholar.google.com/scholar?hl=en&q=magnesium+neuroprotective+OR+neurotoxic+OR+ischemia+OR+neurodegenerative). In that search, there's one of the articles (Harkema et al., 1992) in which researchers have discussed the use of parenteral MgATP(2-) to protect against different kinds of trauma. If only someone could market a simple acylated prodrug of ATP (or the adenosine prodrug along with dibasic orthophosphate in a 1:3 ratio or something) that would release adenosine slowly enough not to produce hypotension but quickly enough to outperform the effects of oral ATP. They could have done it back in the 1960's, when researchers were obtaining the first use patents for acylated nucleosides. Think of the effects that type of simple approach (or a prodrug of inosine, etc.) could have had in clinical neuroscience, even in the years since 1992. It's moving up on 20 years since 1992. It's sadly ironic, in my mind, that it's ATP and nucleotides that have all of these effects, that have been researched heavily, and that aren't being utilized and probably won't be for a long time, in spite of the thousands of articles on all of these things. But the irony is that researchers have been dumping nucleotide triphosphates into PCR machines all over the place and have been using them in experiments like candy or "hamburger helper." In any case, no one would think magnesium could be a standalone treatment for strokes or anything, and there are all of those details, as discussed in past postings, related to the fact that magnesium supplementation tends to decrease serum phosphate levels, sometimes very significantly. But those issues are not all that difficult to address, as long as one is aware of the potentially-large magnitude of the interaction, etc.

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.

Heterogeneous Precipitation/Nucleation as a Mechanism Leading to "Chaos" in Magnesium and Phosphate Homeostasis

In their in vitro experiments at physiological pH values, Sheikh et al. (1989) [Shiekh et al., 1989: (http://www.pubmedcentral.nih.gov/picrender.fcgi?pmid=2910921&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/2910921)] found that calcium (Ca2+) acetate was more effective in binding phosphate (Pi) than calcium carbonate was, and the authors also found that magnesium (Mg2+) was less effective than calcium in binding phosphate in vitro. However, Spiegel et al. (2007) [Spiegel et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17971314)] cited research (reference 10, cited on p. 421) in which the authors had made the argument that Mg2+ is likely to bind more phosphate than Ca2+ in vivo, primarily because less Mg2+ is going to be absorbed than Ca2+. I think that argument is likely to be valid, and the main thing would be to try to separate the administration of supplemental Mg2+ from the administration of Pi by at least 2 hours [Heaney, 2004: (http://www.mayoclinicproceedings.com/content/79/1/91.full.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/14708952)]. But the significance of the in vitro comparison of Ca acetate and Ca carbonate is that those experiments (Shiekh et al., 1989) provide an indirect explanation of one mechanism by which so-called Pi binders, including Ca alpha-ketoglutarate, reduce serum Pi in vivo. The mechanism is the formation, in the intestinal lumen, of heterogeneous precipitates (a.k.a. epitaxial growth of precipitates, heterotopic crystallization, etc.) that are amorphous or crystalline in their structures and that are composed of one or more anionic species, including urate or oxalate or ketoglutarate or other dicarboxylic acids (or even unconjugated bilirubin or bile salts, etc.) and Ca or Mg or both [(http://scholar.google.com/scholar?hl=en&q=epitaxial+phosphate+calcium)]. I should mention that the sequestration of Pi in the intestinal tract can't explain the reductions in serum Pi that the parenteral administration of amino acids has sometimes produced (http://hardcorephysiologyfun.blogspot.com/2009/09/reductions-in-serum-phosphate-induced.html). The reason is that the amino acids were administered parenterally and not enterally (i.e. jejunally or duodenally or orally or whatever variation on that). In any case, these are some of the articles showing the serum Pi-lowering effect of Ca alpha-ketoglutarate [Birck et al., 1999: (http://ndt.oxfordjournals.org/cgi/reprint/14/6/1475.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/10383011); (http://scholar.google.com/scholar?hl=en&q=calcium+ketoglutarate+phosphate+binder)], and I don't think much of those calcium salts of organic anions as Pi binders or of Ca supplements in general. But it's interesting that bilirubin can form heterogeneous precipitates with Ca Pi, and "Ca Pi" supplementation decreased plasma bilirubin without altering the rates of urinary calcium or phosphate excretion [Van der Veere et al., 1997: (http://www.ncbi.nlm.nih.gov/pubmed/9024299)]. These are some other articles that show that effect (http://scholar.google.com/scholar?hl=en&q=calcium+phosphate+bilirubin).

Those mechanisms could mean that reasonable but not excessive intakes of Pi could serve to increase or "maintain" the excretion of bilirubin, but higher dosages could produce more of a nucleating effect and produce cholelithiasis (gallstones composed of mixed Ca and Mg precipitates of urate and phosphate, etc.). In the context of purine nucleotide supplementation, small changes in the ratios of phosphate, derived from nucleotide monophosphates or triphosphates, to exogenous-nucleotide-derived urate and xanthine could influence the formation of those types of precipitates. Adenosine that reaches the liver could increase Pi uptake by sequestering Pi in purine nucleotides and by increasing the activities of phosphofructokinase and other glycolytic enzymes, but an increase in biliary urate excretion (a significant amount can be excreted in the bile, rather than the urine), as a result, could increase the formation of heterogeneous precipitates with Ca Pi in the common bile duct and cause a biliary obstruction, etc. Those types of interactions would probably not be significant at most dosages, in my opinion, but it's potentially useful to be aware of that type of thing. That type of "extreme" scenario would be unlikely in anyone who is using reasonable dosages but could be more likely to occur in a person who is diabetic or insulin-resistant, for example.

It's interesting that the absorption of Mg, in particular, can also be drastically decreased by its binding to and sequestration by bile acids and unabsorbed fatty acids in people who display malabsorption due to liver disease, etc., and I've cited research on that in past postings. The dosages of Mg that researchers had to use to overcome that binding effect and just correct the deficiency state, in children who were undergoing treatment for liver disease, works out to a dose of 2380 mg/day of Mg for a 70-kg human [Heubi et al., 1997: (http://www.ncbi.nlm.nih.gov/pubmed/9285381); (http://hardcorephysiologyfun.blogspot.com/2009/01/articles-on-pantothenic-acid-vitamin-b5.html)]. That means that most of that 2,380 mg (or equivalent dosages in children) was not even available for absorption. And those children weren't even having to consider the binding of Mg by phosphate, etc. There's actually a scaling factor of about 2 that's sometimes used to convert children's dosages to adults' dosages, but I think that scaling factor is only applicable to children within a fairly narrow range of ages. But even supposing it's 1,190 mg of Mg that's being bound by endogenous bile salts and dietary fatty acids in an adult who has liver disease, it's relevant that as many as 20-30 percent of Americans display some degree of nonalcoholic fatty liver disease. A gram of phosphate can bind up to 1800 mg of Mg. That means the intake of a person who is not taking supplemental Mg and who is adding a gram of phosphate to his or her diet, through meat intake or some other route, could conceivably be receiving a daily Mg intake of "negative 2690 mg," assuming the person gets the usual, measly 300 mg/day from foods. In reality, the Pi wouldn't bind that much Mg in vivo, especially if the Mg were taken at a different time. But the point is that the magnitude of the Mg binding can be very large, and the nucleating effect of some of these endogenous, anionic compounds could create complex dose-response relationships for something like Pi. The formation of heterogeneous precipitates of bilirubin and calcium phosphate could also explain the apparent "phosphate-sparing" effect of calcium phosphate, even though calcium phosphate is more or less insoluble (see Heaney, 2004). (The calcium phosphate could remain insoluble and promote the nucleation of complexes of calcium and bilirubin, thereby reducing the amount of calcium that would be available to bind to dietary phosphate. That could increase the amount of phosphate that would be available for absorption. I don't quite understand the stoichiometries of the binding of soluble calcium with bilirubin and calcium phosphate or magnesium phosphate to form insoluble, heterogeneous precipitates, but it's likely that no one understands those issues.)

Potential Interactions of Urate and Inorganic Phosphate with Xenobiotic Substrates and Physiological Substrates of Organic Anion Transporters

One thing I was going to mention is that an excessive intake of inorganic phosphate, alone or in combination with oral purine nucleotides, could conceivably interact with prescription or nonprescription drugs that are substrates of organic anion transporters (OAT's) or multidrug resistance (MDR) protein transporters. The main types of drug-drug interactions that are given attention in the literature are the interactions that involve the noncompetitive inhibition, induction, or competitive inhibition of cytochrome P450 enzymes. But another type of interaction that would be more difficult to predict or even measure could be the competition of two substrates for export, across the canalicular, or apical, membranes of biliary epithelial cells, into the bile. Urate and phosphate can compete for export into the blood or bile by OAT's on the plasma membranes of different cell types in the liver, and bilirubin (http://scholar.google.com/scholar?q=bilirubin+%22organic+anion%22&hl=en), bile acids (http://scholar.google.com/scholar?hl=en&q=%22bile+acids%22+%22organic+anion%22), and other compounds are also substrates of various OAT's. A lot of different drugs are also substrates of OAT's (http://scholar.google.com/scholar?q=drugs+transport+%22organic+anion%22&hl=en) and might compete with urate or phosphate or xanthine, for example, conceivably, for export into the bile. It's unlikely that these interactions would be significant, in my opinion, except at high or excessive dosages of uricogenic purines or inorganic phosphate or in people who have liver or kidney disease. As I've mentioned in past postings, however, some neuraminidase inhibitors and other drugs or metabolites of drugs that are excreted unchanged or otherwise eliminated primarily by renal excretion might interact more significantly with high dosages of oral purines or with excessive amounts of inorganic phosphate. The effect that could conceivably be problematic would be a slowing, in response to an increase in intracellular urate or phosphate, etc., of the rate of biliary or renal excretion of a given drug. That's one reason it's always necessary to discuss these things with one's doctor.

Nonetheless, urate has been used to treat various forms of liver disease in animal models [one example: Garcia-Ruiz et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16941682)], and researchers have shown that urate can protect against mitochondrial dysfunction induced by a wide variety of treatments that produce mitochondrial dysfunction by increasing peroxynitrite formation (http://scholar.google.com/scholar?hl=en&q=mitochondrial+peroxynitrite+urate+OR+uric). A lot of factors and disease states can increase peroxynitrite formation, and the "antioxidant" or "nitrosative-degradation-by-proxy," more accurately, effects of urate, along with its apparent capacity to decrease or directly inhibit PARP-1 activity, make it more useful than many other compounds or antioxidants, in my opinion. As I've discussed in past postings, it may well be advantageous for an antioxidant, such as urate, to not be regenerated. Nonetheless, urate can, for example, regenerate melatonin and guanosine radical species by apparently-nonenzymatic mechanisms (http://scholar.google.com/scholar?hl=en&q=melatonin+regeneration+urate). And, as far as the rest of this posting is concerned, there's evidence that hypophosphatemia and intracellular phosphate depletion in the liver may contribute to liver damage in some cases and disease states (see past postings). One of the most important considerations in the context of phosphate homeostasis is to be aware that, in my opinion, the "phosphate" contained in inositol hexakisphosphate and other phytate compounds, in cereal grains and "plant proteins," etc., is unlikely to provide much, if any, utilizable phosphate in humans [see here: (http://hardcorephysiologyfun.blogspot.com/2009/08/phytates-as-potentially-poor-sources-of.html); (http://hardcorephysiologyfun.blogspot.com/2009/07/phytates-inositol-hexaphosphate-and.html)]. As far as my own calculation of my "dietary phosphate" intake went, I didn't even bother to include a contribution of cereal-grain phosphate. I put a big "NOTH-THING" by the spot on the page for the mg phosphate derived from phytate-containing foods. But I can't make that determination or calculation for anyone except myself. If I had been in the business of obtaining "hocus-pocus-microbial-phytase-derived-phantom-phosphate" phosphate from foods, maybe I'd have listed an actual number. But anyway, as with any compound, bizzarely-high dosages could cause problems. In response to massive dosages of either uricogenic purines or inorganic phosphate, those problems could take the form of interactions with other OAT substrates.

Crucially-Important and Mind-Bending Interactions in Phosphate and Magnesium Homeostasis

The authors of these articles [Thumfart et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18701629); Wei et al., 2006: (http://www.pdiconnect.com/cgi/reprint/26/3/366.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/16722031)] have discussed the evidence that serum magnesium (Mg) levels tend to be inversely correlated with serum parathyroid hormone (PTH) levels, and Wei et al. (2006) discussed all of the evidence that magnesium repletion can be protective against calcification and thrombosis and markers of cardiovascular disease. The inverse relationship between the serum Mg and PTH levels is not widely known in the literature, and I've only recently even seen research on it. It's really important, and Mg is just really important, in general, in my opinion. Most of the research and articles on Mg and PTH have focused on the hypocalcemic hypoparathyroidism that can occur in severe Mg deficiency, but repletion of Mg in severely-deficient animals or humans only increases serum calcium (Ca) back to normal levels, by increasing (restoring) the normal capacity of the parathyroid glands to release PTH in response to decreases in serum Ca. At serum Mg levels or dietary Mg supplies that are higher than those that are required for those most basic functions, Mg is thought to suppress PTH levels by acting as a "weak" activator, like a partial agonist, almost, of the calcium-sensing receptor(s) that mediate the suppression of PTH release in response to increases in serum Ca (Thumfart et al., 2008). Paradoxically, Mg can also increase urinary calcium excretion by reducing the reabsorption of calcium in the renal tubules (Thumfart et al., 2008). It's important to note that that increase in urinary Ca excretion would be likely to occur in conjunction with the decreases in the risk of nephrocalcinosis that researchers have generally found in response to increases in Mg intake. In the case of phosphate (Pi) repletion, the decreases in urinary Ca excretion are thought to result from the suppression of PTH-mediated bone resorption. Thus, Pi is thought to actually reduce the amount of Ca that is filtered in the glomeruli, and Mg may partially act by inhibiting Ca reabsorption in the renal tubules (proximal tubules and distal tubules). But the research I cited above suggests that Mg can increase the rate of urinary Ca excretion and even decrease the serum Ca levels and exert a concomitant, suppressive effect on PTH release. That's a really unusual set of effects. Pi can decrease serum Ca (an effect that is probably undesirable) and decrease urinary calcium excretion but can also elevate PTH levels, and that's an effect that could be attenuated, for better or worse, by an increase in Mg availability to the parathyroid glands or the Ca sensing proteins in the renal tubules, etc. Additionally, many of the bizarre derangements in the homeostatic regulation of Ca and Pi that have been found in response to long-term, excessive Pi supplementation could result, in some cases, from Mg depletion. I also get the general sense that a "high" Pi intake will tend to produce plasma volume expansion and lead to a reduction in urinary sodium excretion, and that could tend to oppose the natriuretic effect that high doses of Mg can sometimes produce. Another way of looking at it would be to say that a high or excessive Pi intake may produce plasma volume expansion by reducing Mg absorption or by increasing Mg turnover by other mechanisms, and Mg has sometimes produced low-level diuretic effects [Walker et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9861593?dopt=Abstract)], by mechanisms that aren't clear.

Incidentally, this is another article that includes a discussion of the antithrombotic effects that increases in Mg availability can produce [Maier et al., 2004: (http://www.ncbi.nlm.nih.gov/pubmed/15158909)], and I've been meaning to collect some of the articles that show the antithrombotic effects of Mg repletion or of elevations in the extracellular Mg levels (the steady-state, extracellular Mg levels are not necessarily or even usually going to be elevated much or at all, even in response to Mg supplementation that increases intracellular Mg levels).

What's really interesting is that low serum Mg and low serum Pi tend to go hand in hand and produce many of the same manifestations, including rhabdomyolysis and decreases in red blood cell (RBC) deformability and hemolytic anemia and decreases in RBC 2,3-DPG and ATP, etc. Oken et al. (1971) [Oken et al., 1971: (http://www.bloodjournal.org/cgi/reprint/38/4/468.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/5571433)] found that Mg deficiency caused hemolytic anemia, reticulocytosis in combination with erythroid hyperplasia in the bone marrow (basically meaning that some erythroid colony-forming units in the bone marrow may be enlarged and hyperresponsive to erythropoietin and that the immature RBC's are more numerous and are also undergoing apoptosis at a high rate, because of Mg depletion), decreases in serum phosphorus (and, hence, serum Pi, also), and decreases in the RBC 2,3-DPG and ATP concentrations and in the overall glycolytic activity in RBC's. Piomelli et al. (1973) [Piomelli et al., 1973: (http://www.bloodjournal.org/cgi/reprint/41/3/451.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/4690142)] also found hemolytic anemia in Mg depleted rats and cited research that had shown hypophosphatemia and hypomagnesemia to occur concomitantly in animals and humans.

It's also really important to note that a lot of articles have shown that Mg supplementation at dosages that would produce "desirable" effects, in my opinion, can decrease serum Pi or produce outright hypophosphatemia. And Pi supplementation can produce hypomagnesemia and intracellular Mg depletion. I think that the amounts of supplemental Mg that might be required to compensate for those effects of Pi repletion could be large and could be too high for many people to easily "accept." But, if the Mg is binding to Pi in the GI tract and precipitating, it's not going to be absorbed (there could be some solubilization in response to pH changes along the GI tract, but, for the most part, the precipitation is going to be permanent and is going to mean that the Mg and Pi are "lost"). One way of considering this would be to say that some percentage of a dose of Pi, from the diet or low-dose supplement, under a doctor's supervision, is going to be absorbed and some percentage is going to not be absorbed and probably bind some amounts of Mg and Ca. The intestinal absorption of Ca and the maintenance of serum Ca and renal Ca reabsorption are much more effectively maintained and regulated, in my opinion. Researchers have noted that the serum Ca is relatively stable, even in terms of circadian changes, than the serum Pi. The serum Pi fluctuates wildly throughout the day and in response to exercise, etc. I think the serum Mg levels are not as unstable as the serum Pi levels are, but the intracellular Mg concentrations are very easily depleted, such as in response to catecholaminergic stimulation, etc. Assuming one is using any supplemental Mg and Pi under a doctor's supervision, there's really a need to not be afraid to increase the supplemental intake of Mg, from Mg salts (such as magnesium hydroxide or magnesium oxide) slowly but relatively freely, to compensate for the reductions in absorption that are likely to result from the extra Pi. It wouldn't be a good idea to increase the Pi as freely as one might increase one's Mg intake, but part of the point of this is that an adequate degree of Mg availability is really obligatory for many of the effects of Pi repletion to be sustained in the longer term. One way to approach this type of problem would be to decide on some dosage of supplemental Mg that is tolerable and safe, under a doctor's supervision, and then to increase the ratio of Pi to Ca, assuming one would want to do this, in the first place. That would allow one to evaluate the effect of the Pi increase, from food or low-dose supplements, with the "knowledge" of the baseline effects that the initial Mg dosage produced. There's still a tendency for a lot of the research to focus on the most severe manifestations of the depletion of Mg or Pi or both (hypophosphatemia or hypomagnesemia), but intracellular Pi and Mg depletion tend to occur long before overt hypomagnesemia and hypophosphatemia occur. In any case, those articles I cited are just the tip of the iceberg. Resistance exercise that is done correctly, for example, can drastically deplete intracellular Mg and Pi concentrations, but the tendency has been to focus, in the case of Pi and RBC 2,3-DPG, on the short-term, post-exercise increases in RBC 2,3-DPG or serum Pi. But the more important issues have to do with the changes that occur in the days after the workout. It doesn't make sense to say that resistance exercise that correctly emphasizes the eccentric movement is going to increase RBC 2,3-DPG in the hours after exercise and then cause those levels and the intracellular Pi levels in skeletal or cardiac myocytes to also remain persistently elevated. Where would the Pi come from. It's likely that intense exercise can produce drastic depletions in intracellular Pi levels, but I haven't seen a lot of data on that. The increases in catecholaminergic transmission during resistance exercise would be expected to produce a significant depletion of intracellular Mg, and this has been shown to occur. And beta-adrenoreceptor agonists can produce hypophosphatemia and hypomagnesemia in the long term, etc. Even lowly L-methylfolate could reasonably be expected to increase Pi and Mg turnover, in my opinion, as a result of its apparent catecholaminergic effects.

Phosphate as a "Magnesium-Binder"

I was looking at the amounts of calcium that can bind to a given amount of dietary phosphate and form an insoluble precipitate [Heaney, 2004: (http://www.mayoclinicproceedings.com/content/79/1/91.full.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/14708952)]. That quantitative relationship is likely to be important in determining the amounts of supplemental magnesium that would, in theory, in my opinion, be required to compensate for the formation of insoluble complexes of magnesium and phosphate (Pi) in the GI tract. Magnesium is probably just as effective as calcium (a recent article shows it to be more "effective" than calcium) as a "phosphate binder" [here are some of the older articles: (http://scholar.google.com/scholar?q=%22Long-term+use+of+magnesium+hydroxide+as+a+phosphate+binder+in+patients+on+hemodialysis%22&hl=en)], but, assuming magnesium and calcium are equipotent as phosphate binders, the quantitative relationship that Heaney (2004) mentioned would mean that 1,000 mg of supplemental phosphate (I'm assuming phosphorus means phosphate, given the convention) could theoretically bind up to 1,827 mg of magnesium (or 3012 mg calcium). In reality, it wouldn't be that high, and the articles on phosphate binding (it's used to treat hyperphosphatemia in people who have renal failure) discuss those discrepancies between in vitro data and in vivo data, etc. And the effect could be minimized through the use of chelated magnesium asparate, assuming one can tolerate it, or by separating the phosphate intake from the magnesium intake or by using organic phosphate compounds, such as ATP or fructose-1,6-diphosphate (if those phosphorylated sugar compounds were actually available). The organic phosphate compounds can be absorbed intact, to some extent, and any complexes formed with magnesium would still be soluble (in all likelihood, in my opinion), meaning that the complex could also be absorbed intact by solvent-drag-facilitated passive diffusion, etc.

But the point is that, in the case of magnesium oxide, there could conceivably be a really significant reduction in the absorption of magnesium. I think it would probably be more pronounced with something like disodium phosphate than with ATP or dietary phosphate. There's a lot of research in this area, and it's really interesting. In any case, it's almost impossible for a person who does not have kidney failure to become hypermagnesemic, and a couple of those articles discussed the use of dosages of up to 3000 mg of magnesium hydroxide, which is about 42 percent elemental magnesium (966-1260 mg elemental Mg), as a phosphate binder in people with renal failure. Those dosages didn't cause hypermagnesemia, even in people with renal failure. But the main concern is not necessarily the potential for hypermagnesemia, in my opinion, but the disturbances in electrolytes or in nerve fiber conduction or in the short-term regulation of blood pressure, etc., in susceptible individuals, and so one would want to discuss these things with one's doctor. The main thing is to be aware of the potentially large magnitude of the "interaction" with magnesium oxide or other magnesium salts. A lot of the magnesium might not be absorbed. That's what I mean when I refer to an "interaction." Chelated magnesium aspartate (this is a chelated form that has been researched a lot and that doesn't provide massive amounts of glycine) is thought to be largely absorbed intact, through dipeptide transporters or passive diffusion, and its solubility in chelated form would probably prevent it from precipitating with phosphate. It's conceivable that there could still be some binding to phosphate or pyrophosphate, but, anyway, "it's a wrap" for tonight: quantitative estimate of the maximal magnesium-binding capacity of phosphate in the intestinal luminal fluid.

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.

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

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

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

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

Depletion of Intracellular Uridine in Response to Intracellular Phosphate Depletion: Potential Relevance to mtDNA & Nuclear DNA Turnover and Repair

In this article [Makras et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18252791)], Makras et al. (2008) described a person who had X-linked hypophosphatemic rickets (XLHR), a genetic disorder that impairs the reabsorption of phosphate, from the tubular fluid, in the proximal tubules, and in whom roughly seven years of phosphate supplementation was ultimately required to completely ameliorate his myopathy (muscle weakness, etc.). The authors noted the mysterious quality of the myopathy and their finding that the severity of the myopathy had generally been independent of the person's serum phosphate levels. The authors also noted that the myopathy had been worsened during periods of vitamin D intoxication. I'm not sure if they're talking about calcitriol or vitamin D, but it probably doesn't matter, to some extent. Hypercalcemia could conceivably result from supplementation with either vitamin D (at the high doses used in patients with XLHR) or calcitriol and could cause excessive calcium influx into myocytes, thereby impairing mitochondrial ATP formation, or cause hypercoagulability, etc.

Although the authors wrote that vitamin D usually causes rapidly-emerging improvements in muscle weakness in people who do not have inherited mutations that affect phosphate homeostasis, as in XLHR, it's conceivable to me that the myopathy could have resulted from mitochondrial dysfunction, perhaps as a result of acquired mitochondrial DNA (mtDNA) mutations, as a consequence of the phosphate depletion. The fact that the degree of muscle weakness was independent of the serum phosphate is not surprising, and the intracellular phosphate levels are known to frequently, if not generally, be independent of steady-state serum phosphate levels in normal humans given low dosages of supplemental phosphate. Given that intracellular phosphate depletion is known to deplete ATP and purine nucleotide pools and that the depletion of the pools of purine deoxyribonucleotides can impair mtDNA replication (see past postings), it's conceivable that intracellular phosphate depletion could impair DNA repair and lead to a gradual accumulation of mtDNA or even nuclear DNA mutations. It's worthwhile to note that the maintenance of an adequate pool of each of the major intracellular purine nucleotides is a prerequisite for the maintenance of pyrimidine salvage. I think some of that has been shown in the context of fructose-induced hepatic ATP depletion. I think researchers have shown that fructose can deplete uridine from the liver and transiently elevate plasma uridine, as one might expect in response to fructose loading. Here are some references on that [see page 33 of the chapter of the book by Davies et al., 1998, who found that the plasma uridine levels increased soon after fructose administration in humans and then decreased a lot by 4 hours after a meal; sounds fantastic: (http://scholar.google.com/scholar?q=fructose+uridine+plasma+OR+serum&hl=en)].

Thus, intracellular phosphate depletion could conceivably contribute to the development of mutations in nuclear DNA and to the development of some of those more severe myopathies or intractable disease states, such as chronic fatigue syndrome, by leading to a depletion of both purines and pyrimidines. That's just my opinion, however. It's noteworthy that DNA repair consumes a lot of ATP, and some authors have suggested, as I noted in my old folic acid paper (see past posting), that the depletion of intracellular total folates might cause apoptotic cell death in neurons by "DNA-repair-associated" ATP depletion. They meant that there would be a futile cycle of DNA damage, in response to folate depletion and increases in the dUMP/dTMP ratio, and DNA repair and that the DNA repair would ultimately consume so much ATP as to lead to apoptotic cell death, such as in response to ischemic episodes or strokes that can cause a lot of DNA damage. Davies et al. (1998) argued that fructose-induced phosphate depletion in the liver had caused both the purine depletion, as evidenced by the elevations in serum uric acid, and the uridine export from the liver. I'm not suggesting that more is always going to be better, and those articles on the overlapping mechanisms governing the efflux of uric acid and inorganic phosphate, as discussed in recent postings, suggest that the metabolic cost or competitive inhibitory effects of excesses of intracellular inorganic phosphate could become significant, past a certain point, and derange the transport of organic anions other than uric acid, etc. Although the research suggests that a lot of phosphate would be required to create that type of state, it's worthwhile to discuss these things with one's doctor.

My view is that the data on the dosages of phosphate used in people with XLHR (and in other genetic disorders that reduce phosphate reabsorption) is relevant to normal humans, with regard to the risk of nephrocalcinosis, but I can think of a number of possible objections to that view. The first would be that, in people with XLHR, the rate of phosphate (Pi) reabsorption would be lower than it would in normal people and that that would decrease the risk, in comparison to normal people, of intracellular calcium phosphate precipitation. I should mention that, in that long review on nephrocalcinosis that I recently discussed, the author noted that calcification can occur either extracellularly (and "luminally" or intraluminally), on the luminal membranes of the proximal or distal tubule cells or in the interstices of the tight junctions, or intracellularly, in the cells of the renal tubules. Thus, one could argue that normal people would have the same risk of intraluminal calcification as people who have XLHR would but that normal people would have a higher risk of intracellular calcification, in response to a given dosage of supplemental phosphate, as people who have XLHR would. In normal people, however, the proximal tubules are able to vary the percent reabsorption to between something like 80 and 99 percent, and that means there would be a lot of potential for the proximal tubules to increase the urinary excretion of phosphate in response to some dosage of supplemental Pi. I just think the risks are basically similar for normal people as they are for people who have XLHR.

The reason I'm focusing on the reabsorption is that XLHR doesn't affect the glomerular filtration of serum phosphate, except to the extent that the cells of people with XLHR might be more "hungry" for phosphate and might clear the serum phosphate, from a dosage of phosphate, more rapidly than a normal person's cells might (thereby producing an indirect decrease in the amounts of phosphate filtered per unit time). In other words, the acute elevations in serum phosphate could conceivably be larger in normal people than in people who have XLHR. But that presupposes that a person has no capacity to tell if some change in his or her phosphate to calcium intake ratio, for example, is producing any benefit. If there's no obvious benefit, presumably there wouldn't be an impetus to continue taking any reasonable amount of phosphate, with the approval of one's doctor. It would also, obviously, be important to spread the dosage out across the day as much as possible and to consider limiting any dosage of supplemental vitamin D to 2000-4000 IU or less, given that hypercalciuria is thought to be a major factor that can increase the risk of nephrocalcinosis. Some authors have suggested splitting the total daily dosage of phosphate, in people who have XLHR, into 8 dosages, spread out across the day, instead of the usual practice of splitting the dosage into 4-5 increments. Another objection I can think of would be that the PHEX protein or the Na(+)/Pi cotransporter might be expressed in myocytes or myogenic satellite cells or some other extrarenal cell type. That could mean that mtDNA replication or some other Pi-sensitive metabolic process would be specifically affected in the muscles and would not be likely to show up in normal people. But I don't see how a pure and severe case of Fanconi's syndrome couldn't produce the same kinds of long-term problems in postmitotic cell types as something like XLHR can. It probably wouldn't take 7 years to treat the problem in a normal person, but I just think that there's a need to think of this type of thing with the long view in mind. It's necessary for someone to do long-term safety research using supplemental phosphate in normal people and to use reasonable amounts of dietary calcium, etc. Or someone could do that type of research in people who have chronic fatigue syndrome. I don't know what the best approach would be. One could argue that reasonable and low dosages of phosphate would improve both purine and pyrimidine salvage and could help limit something like the age-associated reductions in mtDNA copy number in different cell types. These are just my off-the-cuff thoughts, but I think the notion that 7 days of "phosphate loading" is enough to make anyone "A-okay," in view of the mechanisms by which both the purine and pyrimidine ribonucleotide pools could become depleted intracellularly, for example, doesn't make a whole lot of sense to me. If the intracellular phosphate depletion is brief, then it makes sense to me that a brief period of time would be required to correct that depletion. But one is not even going to be able to tell if the intracellular phosphate levels are being maintained in some cases, given the frequently-observed independence of the intracellular and extracellular phosphate concentrations. So someone would have to do muscle biopsies or use 31P-MRS intermittently or measure red blood cell 2,3-diphosphoglycerate levels as a surrogate for the measurement of the intracellular Pi levels in myocytes, etc.

Potential for Competition Among Phosphate, Uric Acid (Urate), and Antivirals Used to Treat Influenza for Transport by Organic Anion Transporters

The authors of this article [Yabuuchi et al., 1998: (http://jpet.aspetjournals.org/cgi/reprint/286/3/1391)(http://www.ncbi.nlm.nih.gov/pubmed/9732402?dopt=Abstract)] describe the capacity of the type I Na(+)/Pi cotransporter (NPT1), a sodium and inorganic phosphate (Pi) transporter, to transport either organic anions, including probenecid, or inorganic phosphate (Pi) out of the liver and into the blood. Yabuuchi et al. (1998) noted that probenecid can compete with Pi for transport by NPT1, and this could conceivably mean that a higher intake of Pi might inhibit the efflux of uric acid (urate, UA), an organic anion whose reabsorption by proximal tubule epithelial cells can be inhibited by probenecid (http://scholar.google.com/scholar?hl=en&q=urate+probenecid), from the liver or otherwise influence the efflux or uptake of urate or xanthine by cells in the liver or kidneys, etc. (http://scholar.google.com/scholar?hl=en&q=%22inorganic+phosphate%22+anion+transporter). It's also conceivable that increases in extracellular or, in a more likely event, intracellular Pi could slow the elimination of antiviral drugs used to treat influenza. For example, Oo et al. (2002) [Oo et al., 2002: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=127254&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/12019123)] noted that the active metabolites of some neuraminidase inhibitors are mostly excreted unchanged, such as through their uptake by the proximal tubule cells, from the peritubular capillaries, and efflux across the luminal (apical) membranes of proximal tubule epithelial cells into the tubular fluid. Karie et al. (2006) [Karie et al., 2006: (http://ndt.oxfordjournals.org/cgi/reprint/21/12/3606.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/16799172)] noted that some neuraminidase inhibitors do not serve as substrates for cytochrome P450 oxidoreductases in the liver and do not inhibit those enzymes either, and that's a major reason that their active metabolites are mostly excreted unchanged by renal tubular excretion. Probenecid competes with some of these These interactions would probably not be very likely and would be most likely to occur, if at all, in people whose kidney function has already been diminished, as a result of age or other factors. This is a hastily-chosen article that describes the capacity of probenecid to inhibit the transport and, hence, renal excretion of some neuraminidase inhibitors or their active metabolites [(http://www.cdc.gov/Mmwr/preview/mmwrhtml/rr4814a1.htm); (http://scholar.google.com/scholar?q=probenecid+neuraminidase+inhibitor&hl=en)], and that basically means that oral purines or phosphate supplementation could conceivably slow the elimination of some neuraminidase inhibitors, and that wouldn't necessarily be desirable. It might sound good, and some people have proposed the use of probenecid to allow for the use of neuraminidase inhibitors at lower dosages (thereby allowing more people to be treated with antivirals, in the event of a "1970's-style shortage" of antivirals). But that could be a dangerous approach, given that the movement and constant efflux of some neuraminidase inhibitors is necessary to prevent the potentially problematic effects of their accumulation intracellularly, in cells in the liver or kidneys.

Thus, if one were taking an antiviral to treat an influenza infection and also taking some oral purine compound or source of inorganic phosphate (Pi), one might need to reduce the dosages of those or, as discussed by Karie et al. (2006), reduce the dosages of the antivirals. It would seem that reducing the dosage of the antiviral would not be the better approach, in theory, but one would obviously want to discuss this with one's doctor. Some of the major old M2 protein inhibitors, used as antivirals in the treatment of influenza, are derivatives of 1-aminoadamantane and are therefore also excreted unchanged. Aminoadamantane derivatives are apparently transported by organic cation transporters and would seem to not compete with UA or phosphate, but probenecid is a weak base and can sometimes inhibit the transport of substrates of organic cation transporters (http://scholar.google.com/scholar?hl=en&q=probenecid+aminoadamantane). There are strange ways in which substrates of organic cation transporters can influence the transport of other substrates (drugs or physiological compounds) of organic anion transporters [Khamdang et al., 2002: (http://jpet.aspetjournals.org/cgi/content/full/303/2/534)(http://www.ncbi.nlm.nih.gov/pubmed/12388633?dopt=Abstract)], maybe because they, like probenecid, are weak bases and could either be protonated or deprotonated or because they contain more than one ionizable group. There can be pH extremes and variations in the tubular fluid, for example, and there could be indirect interactions. An increase in the reabsorption of UA could, for example, be pH dependent and thereby produce an indirect, pH-sensitive reduction in the excretion of a drug that UA, by its binding to an efflux transporter intracellularly, in proximal tubule cells, and relative failure to serve as a substrate for transport by that transporter, competes with for transport, etc.

Another implication is that increases in the intracellular Pi concentration could reduce the loss of purine nucleotides both by inhibiting adenosine deaminase (and by activating adenosine kinase, arguably) and by reducing the efflux of cAMP or cGMP or other purine substrates of some organic anion transporters or multidrug resistance proteins that transport purines out of cells. This might mean that phosphate could, apart from its role in promoting normal purine salvage, serve as a dose-reducing agent for oral purines, such as ATP disodium, even in the absence of an influenza infection, obviously. But that's more theoretical, and these are just my opinions. Obviously, other medications, including but not limited to some antibiotics, are transported by organic anion transporters, too, and that's another reason one should discuss this type of thing with one's doctor.

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

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

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

Reductions in Serum Phosphate Induced by Supplementation With Free-Form Amino Acids: Interactions of Phosphate Homeostasis with Renal Ammoniagenesis

These articles [Heidland et al., 1978: (http://www.ajcn.org/cgi/reprint/31/10/1784.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/707333); Lamiell et al., 1990: (http://www.ncbi.nlm.nih.gov/pubmed/2108005)] show that supplementation with free-form amino acids, particularly in conjunction with a low- or "no-phosphate" diet (Lamiell et al., 1990), can fairly drastically reduce serum phosphate and also increase serum calcium. I've discussed some of these articles in past postings (http://hardcorephysiologyfun.blogspot.com/2009/02/potential-for-hypophosphatemia-or.html). Heidland et al. (1978) found that the serum calcium levels varied inversely with the serum phosphorus levels, in the people who had been given supplemental essential amino acids, and the authors suggested that that inverse relationship might have resulted from the increases in serum 1alpha,25-dihydroxyvitamin D3 (calcitriol, hormonal vitamin D, HVD) and the resorptive effect of an increase in serum HVD (?). These are really good articles, and I've never seen anyone mention that increase in "calcium mobilization from bone" (Heidland et al., 1978, p. 1791) in response to an increase in serum HVD. It's not widely-recognized, but there is a lot of research showing that increases in serum HVD can produce effects that are basically opposite to those of increases in autocrine or paracrine HVD (HVD that is formed in response to increases in extracellular 25-hydroxyvitamin D and that acts on nearby cells or in the same cell in which it is formed), and those paradoxical effects seem to show up more in relation to the calcemic or calcium-transport-modifying effects of HVD. For example, increases in HVD are known to be somewhat permissive with respect to soft-tissue calcification in animals, but increases in 25-hydroxyvitamin D, in the absence of concomitant increases in serum calcium (effects that are potentially more likely to occur in response to UV-induced increases in vitamin D than in response to oral vitamin D, in my opinion, given that oral vitamin D is likely to be more calcemic, etc.), have generally not increased soft tissue calcification and may have the potential to decrease soft-tissue calcification by, in theory, helping to prevent the osteoblastic differentiation of smooth muscle cells, etc. But combining high-dose vitamin D3 with high-dose calcium supplements has the potential to cause problems, and Heidland et al. (1978) suggested that the association of the amino acid supplementation with hypercalcemia, in some people, had been a result of the extra calcium supplementation (coupled with the relative absence of dietary phosphate).

A lot of these articles on these strategies for managing hyperparathyroidism and hyperphosphatemia in people who have kidney failure only look at serum phosphate or serum parathyroid hormone (PTH) levels and don't consider the levels of intracellular phosphate or the context in which the increases in PTH levels are occurring, and these are problematic aspects of a lot of these articles (I'm not talking about the ones I cited above). Lamiell et al. (1990) found, for example, that, after the second administration of the zero-phosphate parenteral nutrition formula was given to a person (they gave it and discontinued it a few times before they determined that hypophosphatemia or intracellular phosphate depletion, in addition to hyperammonemia, was causing the encephalopathy, and this is understandable), the encephalopathy occurred when the serum phosphate was normal. Lamiell et al. (1990) attributed that encephalopathic episode to hyperammonemia, and that's plausible, but it's important to remember that the intracellular inorganic and organic (i.e. ATP and ADP, phosphocreatine, etc.) phosphate levels can be significantly depleted in a person whose steady-state serum inorganic phosphate levels are normal [for example, Ambuhl et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10561144)]. Many most articles whose authors have measured the intracellular 2,3-diphosphoglycerate (2,3-DPG) concentrations in red blood cells have shown that have shown that those concentrations don't correlate at all with serum phosphate levels. I also think there's a danger in thinking that these derangements in calcium and phosphate homeostasis will only occur in people who have renal failure, but supplemental phosphate decreased urinary calcium excretion in normal people who had evidently not been exhibiting hypercalciuria [Heaney and Recker, 1987: (http://www.ncbi.nlm.nih.gov/pubmed/2851341)]. So some of these unspoken assumptions, such as the assumption that massive doses of calcium are going to not cause hypercalcemia or pathological effects in people who do not have kidney disease, in short-term trials with calcium supplements have the potential to be invalid.

The mechanisms by which free-form amino acids can decrease serum phosphate are not well-understood, but Heidland et al. (1978) noted that the persistence of the decreases in serum phosphate over the long term, in some people who are given supplemental amino acids, argues against the idea that the hypophosphatemic effect is a result of the "refeeding syndrome" or even something akin to it. I think it's caused by changes in the interactions of renal ammoniagenesis with the mechanisms governing phosphate reabsorption in the proximal tubules, and those interactions basically boil down to changes in acid-base homeostasis. For example, the acute, supplemental-glutamine-induced increases in serum bicarbonate that can occur in humans could be expected to favor an increase in phosphate transport into cells. There probably is an increase in phosphate uptake into cells, in response to some amino acids, but it's noteworthy that excessive or high doses of some mixtures of free-form amino acids seem to be more likely to produce hyperammonemia than protein does. Lamiell et al. (1990) attributed that to the absence of arginine in some mixtures, and that's conceivable. But it might be a result of the kinetics of the absorption of free-form amino acids. Their absorption is going to be much more rapid than the absorption of protein-derived amino acids, and that could overwhelm the liver's capacity for ureagenesis. It's also possible that increases in phosphate utilization (or loss of phosphate in the urine, induced by the amino acids) decreases the availability of intracellular inorganic and organic phosphate in a way that impairs the activities of hepatic urea cycle enzymes. But one possibility that seems plausible to me is that amino acids increase urinary phosphate loss and that the loss of phosphate increases renal ammoniagenesis to a degree that is significant enough to disturb the systemic acid-base homeostatic mechanisms and actually contribute to hyperammonemia. Under some circumstances, the overall urea cycle activity is thought to be an important factor in influencing systemic acid-base homeostasis, and Haussinger et al. (1990) [cited and discussed here: (http://hardcorephysiologyfun.blogspot.com/2009/02/urea-cycle-renal-glutaminase-activity.html); Haussinger et al., 1990: ([Haussinger et al., 1990: (http://www.springerlink.com/content/l2vx314521367706/)] basically found that even mild liver dysfunction was associated with a failure of renal ammoniagenesis to downregulate in response to systemic metabolic alkalosis. So, in a normal person, glutamine supplementation might acutely increase serum bicarbonate or only increase it on some days, such as on a day of intense exercise (in response to acidosis), but massive doses of glutamine or other essential amino acids that increase renal glutamine availability (glutaminase and the glutamine cycle are important in the regulation of renal ammoniagenesis, in response to metabolic acidosis) could contribute to the persistently-alkalotic state that can occur even in compensated liver disease, as discussed by Haussinger et al. (1990).

It's worth noting that alkalosis doesn't just increase phosphate uptake into cells (it tends to decrease intracellular phosphate availability in the long-term or even short term, because the uptake into cells provides phosphate to some cells at the expense of others, given the usual, alkalosis-induced reduction in serum phosphate) but impairs the unloading of oxygen from hemoglobin in a "2,3-DPG-depletion-independent" manner. That's one reason why bicarbonate administration can be so disastrous in a person who is hypophosphatemic, as discussed in past postings. One would think that the alkalosis-induced uptake (it's thought to partially be a result of the alkalosis-induced activation of glycolytic enzymes) of phosphate would increase 2,3-DPG levels, but it tends to not be the case in hypophosphatemia. It might be that more 2,3-DPG is driven into skeletal muscle myocytes or other cells that are not red blood cells, or it might be that the 2,3-DPG-independent "impairment" in the unloading of oxygen from hemoglobin tends to offset any potential for an increase in 2,3-DPG formation to occur in response to the alkalosis-induced increase in phosphate uptake by red blood cells. In any case, an inappropriate and persistent increase in renal ammoniagenesis could cause an ammonia-mediated impairment of TCA cycle activity in proximal tubule cells, given that ammonia excesses are known to inhibit TCA cycle enzymes and other mitochondrial enzymes and to interfere with energy metabolism by all sorts of mechanisms. That metabolic toxicity could reduce phosphate reabsorption by proximal tubule cells and exacerbate phosphaturia. Some of these articles by Ambuhl and colleagues (http://scholar.google.com/scholar?hl=en&q=ambuhl+phosphate) look promising as sources of information on those types of mechanisms.

Effects of Acidic vs. Neutral vs. Alkaline Phosphate Preparations on Urinary Calcium Excretion: Interactions With B6 & Glutamine & Insulin Metabolism

These articles [Thomas, 1978: (http://www.ncbi.nlm.nih.gov/pubmed/351267); Lau et al., 1979: (http://www.ncbi.nlm.nih.gov/pubmed/44888)] are really good, and Lau et al. (1979) found that the administration of alkaline phosphate preparations [salts of HPO4(2-)] to people who had hypercalciuria, due to either inappropriately-increased intestinal calcium absorption (absorptive hypercalciuria) or renal hypercalciuria (due to some problem originating in the kidneys and causing an excess of urinary calcium excretion), reduced the rate of urinary calcium excretion much more than neutral phosphate preparations or reductions in the subjects' dietary calcium intakes did. Thomas (1978) also noted that alkaline phosphate preparations but not acidic phosphates reduce urinary calcium and do only (or, probably, mainly) in people who are hypercalciuric. Lau et al. (1979) and Thomas (1978) also cited and discussed research showing that oral phosphate preparations increase pyrophosphate excretion and probably inhibit renal calcifications and stone formation, in part, by that mechanism. I don't know what the mechanism is thought to be for that effect, but it's conceivable that it occurs because inorganic phosphate, at concentrations found in vivo, can inhibit alkaline phosphatase activity (a high smooth-muscle-cell alkaline phosphatase activity tends to increase the risk of soft-tissue calcification by breaking down an inhibitor of calcification, namely pyrophosphate, into orthophosphate [HPO4(2-)], which can, at excessive concentrations, contribute to calcification) [Coburn et al., 1998: (http://jcem.endojournals.org/cgi/content/full/83/11/3951)(http://www.ncbi.nlm.nih.gov/pubmed/9814474?dopt=Abstract)]. A low alkaline phosphatase activity on the plasma membranes of various cell types or even in the serum could conceivably increase intracellular PLP, or coenzymated vitamin B6, and reduce extracellular or serum PLP levels, given that some minimal degree of alkaline phosphatase activity is required for cleavage of PLP and, hence, entry of B6 into cells, where it's rephosphorylated. So an excess of phosphate could conceivably cause B6 depletion by impairing B6 entry into cells, but Coburn et al. (1998) noted that phosphate depletion is more likely to reduce (intracellular) PLP levels by reducing the availability of phosphate for the phosphorylation of pyridoxal. It's not a technical statement and wouldn't necessarily be true, but maybe adequate amounts of dietary phosphate increase the ratio of pyrophosphate to orthophosphate in the extracellular fluid, etc. Many articles have shown that phosphate depletion tends to be accompanied by elevated serum alkaline phosphatase activity (http://scholar.google.com/scholar?hl=en&q=hypophosphatemia+%22alkaline+phosphatase%22), and that or tissue-restricted increases in alkaline phosphatase activity could occur in response to dietary phosphate depletion and, up to some point, above which the opposite effect could occur, increase the risk of soft-tissue calcification. I think there's some middle ground. The orthophosphates joined together in pyrophosphate have to come from somewhere. Also, Lau et al. (1979) found that neutral phosphate, in comparison to acidic phosphate [salts of H2PO4(-)], caused a lower rate of net acid excretion in the urine (and, not surprisingly, given that a major acidic species in urine is ammonium, caused a lower rate of ammonium excretion) and a higher rate of urinary citrate excretion. Urinary citrate is thought to protect against calcium phosphate or oxalate precipitation. One way of looking at the difference in acid excretion would be to say that more HPO4(2-) might have been available for protonation and excretion as H2PO4(-) in the group supplemented with neutral phosphate than in the acid phosphate group, thereby reducing renal ammoniagenesis and acid excretion as the ammonium ion [NH4(+)], but that's probably an overly-simplistic explanation.

Lewandowski and Rogers (2004) [Lewandowski and Rogers, 2004: (http://www.ncbi.nlm.nih.gov/pubmed/15232796)] found that 100 mg/day of vitamin B6 reduced urinary calcium, phosphorus (phosphate), and the amount of brushite "supersaturation" in relation to other forms of calcium phosphate crystals or amorphous aggregations. They also found that glutamine supplementation reduced "relative calcium oxalate supersaturation" in the urine, implying that it could help to reduce calcium oxalate stone formation, in theory. Maybe glutamine increased citrate excretion by acting as a precursor to citrate, given that researchers have shown that glutamine can increase the levels of intracellular and intramitochondrial citrate and other TCA cycle intermediates in various cell types. A higher urinary citrate is, as Lau et al. (1979) and others have noted, thought to be protective against calcium stone formation, and Cupisti et al. (2007) [Cupisti et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17184967)] found that insulin resistance was associated with a lower rate of urinary citrate excretion. I think 100 mg/day of vitamin B6 is too high as a long-term dosage of vitamin B6, mainly because of the risk of peripheral neuropathy, and 25-50 mg/day would, in my opinion, be a safer dose, in many cases, in the long term. The authors noted that vitamin B6 could have reduced urinary oxalate by increasing its metabolism (by PLP-dependent enzymes), and the authors didn't know why vitamin B6 had decreased urinary calcium excretion. It's mysterious, and I've discussed past research showing that vitamin B6 supplementation, in animals, can decrease serum calcium levels. That's not desirable, necessarily, when one looks at the many different effects, taken together, of vitamin B6. But there's probably some lower dosage that would be safer in the long term and that might produce some of the reductions in calcification of soft tissues (in addition to the antihypercalciuric effect that it could potentially have) that have also been associated with vitamin B6 supplementation (http://scholar.google.com/scholar?hl=en&q=calcification+%22vitamin+B6%22+OR+pyridoxine). Some of those articles are research on the use of magnesium to prevent calcification, and the authors appear to have just mentioned vitamin B6 or used it in combination with magnesium (MgO is magnesium oxide). Some authors have noted that excessive amounts of dietary phosphate could bind to calcium and thereby indirectly enhance urinary oxalate excretion, given that less oxalate in foods would bind to calcium in the gastrointestinal tract (more would be absorbed and then excreted in the urine). That could be important to remember, but who knows how much oxalate comes from foods or if the problem is more that the oxalate isn't being metabolized (or maybe too much is being formed, as a result of the metabolic syndrome, as suggested by the research of Cupisti et al. (2007), cited above). Sayer et al. (2004) [Sayer et al., 2004: (http://cs.portlandpress.com/cs/106/0549/1060549.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/15027893)] was basically that, following some initial calcium oxalate precipitation (crystal formation), calcium phosphate can be more likely to precipitate and form mixed crystals, I guess, through "heterologous nucleation" (this must mean that there's a seeding effect of the calcium oxalate). Thus, vitamin B6 or glutamine (or whatever other factors that could reduce urinary oxalate supersaturation) could conceivably reduce urinary calcium phosphate deposition, in my opinion. Glutamine is probably not going to produce especially reliable effects, but I just think increases in glutamine availability (or maintenance of its availability) interacts favorably with the metabolic effects of inorganic phosphate, by multiple mechanisms.