More on the Potential Relevance of Adenosine Monophosphate to the Adjunctive Management of Depression: Competition Among Nutrients for Excretion

I was just going to briefly discuss some issues that could be relevant, in my opinion, to the potential for the use of purine nucleotides and L-methylfolate, a reduced folate that is available by prescription (with the brand name of Deplin) or over-the-counter (as L-methylfolate, with the trade name of Metafolin, etc.), as adjunctive strategies, used under a doctor's supervision, for dealing with depression. The most basic thing is that the use of adenosine 5'-monophosphate (AMP) is likely to allow for more flexibility in dosing, over the long term, than the use of adenosine 5'-triphosphate (ATP) disodium is, primarily because the hydrolysis of ATP, by a variety of ectonucleotidase enzymes (and also intracellular nucleotidase enzymes, in the intestinal epithelial cells) and esterase enzymes in the luminal fluid of the small intestine, supplies significant amounts of inorganic phosphate. I don't like to mention brand names on here, and I have no financial ties whatsoever to any supplement or pharmaceutical manufacturer or to any other company or commercial interest. The Bluebonnet nucleotide complex is, in my opinion, still the only over-the-counter supplemental source of AMP that provides a meaningful amount of AMP [(http://www.google.com/products?q=nucleotide+bluebonnet&hl=en&aq=f)]. The phosphate is likely to be absorbed more rapidly than the phosphate that is derived from the hydrolysis or, rather, phosphorolysis of polyphosphates that are present in meats, for example. In my most recent postings on the nutritional aspects of phosphate, I was trying to convey that the use of supplemental sources of phosphate has the potential to be problematic, especially but not only when the supplementation continues for more than a few weeks. And I think that supplementing with any source of phosphate without close supervision by a doctor (this would be of absolutely crucial importance and would include the use of blood tests, every couple of weeks, to monitor serum phosphorus, and the use of urinary phosphorus tests to monitor urinary phosphate excretion) has the potential to be extremely hazardous. Sources of supplemental phosphate can elevate serum phosphorus in very unpredictable ways, and the key point is that AMP provides one third as much phosphate as ATP does. Some of the articles I cited in past postings show that the serum phosphorus levels can rise slowly, over many months, and then suddenly increase exponentially, with potentially devastating consequences to kidney function or to the functioning of any tissue, in which the precipitation of calcium phosphate can cause serious damage. This includes any tissue in the body. So I wanted to reiterate and strengthen my warnings about the significant potential for danger that can accompany any use, whatsoever, of a source of supplemental phosphate. Some ATP disodium supplements also contain calcium phosphate binders and silicates, and these excipients can supply significant amounts of calcium that could, in my opinion, either prevent or augment the adverse effects that excessive phosphate intakes can produce, and the effect (prevention or augmentation) would be expected to depend on the dosage and the timing of the administration/intake of the excipient. Messing around with that stuff and worrying about supplements that are loaded up with calcium doesn't sound very good to me, for reasons I've discussed, at length, in the past.

Part of the point with this is that the use of supplemental gluconate, glutamine, creatine, citrate, or adenosine or inosine or other supplements that supply organic anion substrates can, in my opinion, significantly limit the amounts of oral AMP that one can tolerate, and this can become very problematic and puzzling. I've mentioned this in the past, but I want to emphasize that it's very important, in my view, to consider the fact that many nutrients and drugs compete with phosphate and with bile acids, for example, for transport by nonspecific organic anion transporters, in the kidneys and liver. In essence, there tends to be a limit to the total supply of "organic anion transporter (OAT) substrates" (these transporters are very numerous and include the ATP-binding cassette family of transporters, and that family of transport proteins includes the multidrug resistance transport proteins, as in mdr1, mdr2, etc.) that one can ingest, and it can become necessary to "decide" which nutrient one wants to supplement with and to limit the dosages of the other OAT substrates. I've been aware of this for a long time, and many articles discuss the crucial importance of this in the context of liver disease or in relation to kidney-related pharmacology. But it took me a long time to realize that, for example, the dosages of magnesium gluconate, creatine, and glutamine that I'd been taking had been causing me to be unable to use dosages of AMP that I'd wanted to use, in relation to antidepressant augmentation, under my doctor's supervision. The dosages of the individual supplements were not high and were actually fairly low (well, I'd been taking 24 grams of glutamine and about 800 mg of creatine monohydrate, but the amounts of gluconate from magnesium gluconate are likely to have been excessively high), but the total "mass" of OAT substrates (this "mass" includes all of the citrate and glutamate and whatnot that glutamine is metabolized into, and it includes creatinine and other degradation products of creatine and also includes the extra phosphate that can accumulate intracellularly, in response to exercise, in a person who's taking creatine, given that the extra creatine phosphate can, in my opinion, tend to "dump" more phosphate during exercise) was high and was causing me to be unable to tolerate enough AMP. I was noticing that my plasma volume or, less specifically, extracellular fluid volume was high, and this was most obvious during exercise. One manifestation of this can be "water retention" that is obvious at the end of a workout, etc. Uric acid (urate) and other purines that are derived from AMP (and from the guanosine 5'-monophosphate in that nucleotide supplement) also compete with phosphate and with bile acids for transport. I've reduced the dosage of glutamine to 4-8 grams per day and reduced the dosage of creatine monohydrate to about 400 or 500 mg per day (I may reduce this further) and have been able to tolerate more AMP, etc. I also stopped taking magnesium gluconate. If one were taking a small dose of magnesium gluconate, this might not be an issue. But what's a small dose. To supply 30 mg of magnesium, one has to take in something like a gram of gluconate (derived from the magnesium gluconate, after one has ingested it).

This is a very important issue, in my opinion, because AMP is, in my view, probably the most "important" over-the-counter compound that could, in my view, be of sigificance in relation to adjunctive antidepressant strategies that researchers could test and that one could discuss with one's doctor. Methylfolate, for example, has the potential, in my view, to gradually contribute to the decreases in the intracellular purine nucleotide levels that can result, in my opinion, from the use of any number of medications that produce any sort of excitatory effect, whatsoever, on neurotransmission. On a personal level, I've found that none of the antidepressant augmentation strategies, such as Deplin (prescription L-methylfolate), has been nearly as useful, in the long term, without the concomitant use of AMP, but that's just been my experience. It's important to remember that the accumulation of uric acid, produced by purine degradation, can become problematic in the long term and could, in my opinion, contribute to soft tissue calcification, and the safe way to go would be to monitor one's urinary uric acid levels and not just to monitor serum uric acid (serum urate) levels with blood tests. The "excitation-induced decrease in purine nucleotide concentrations" in neurons and astrocytes is a general phenomenon and could even occur in response to the pathological, stress-induced increases in the firing rates of noradrenergic neurons in the locus ceruleus or other adrenergic cell groups (i.e. the A1, A2, and A3-A6 and C1, etc., adrenergic cell groups in different parts of the medulla oblongata). I'll try to put up some better references, but here are some of the countless articles discussing the neuronal-excitation-induced increases in extracellular fluid adenosine levels [Latini and Pedata, 2001: (http://www.blackwellpublishing.com/specialarticles/jnc607.pdf); Rosenberg and Li, 1995: (http://www.ncbi.nlm.nih.gov/pubmed/8548307); Schubert et al., 1997: (http://www.ncbi.nlm.nih.gov/pubmed/9369970); Matsumoto et al., 1982: (http://www.ncbi.nlm.nih.gov/pubmed/1352728); Hagberg et al., 1987: (http://www.ncbi.nlm.nih.gov/pubmed/3585332); Frenguelli et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17459147)(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1920548/pdf/jnc0101-1400.pdf); Hagberg et al., 1986: (http://www.ncbi.nlm.nih.gov/pubmed/2875423)]. When there's the kind of excessive noradrenergic transmission that can lead to the derangements and "decreases" in noradrenaline (norepinephrine) "levels" that have been associated with the worsening of depression or of anxiety disorders, there is the distinct potential, in my opinion, for increases in glutamate release, by glutamatergic neurons that provide synaptic inputs to the noradrenergic or dopaminergic or serotonergic neurons, to also occur and to have the potential to contribute to the excessive increases, as can be induced by stress, in the firing rates of the noradrenergic neurons or other groups of neurons.

The point is that, in my opinion, exogenous AMP, for example, has the potential to replenish some of these losses, even if the effect is not as great as one would like it to be (as it would be in response to intravenous inosine, for example, or an adenosine prodrug or source of intravenous adenosine that would release adenosine very, very slowly into the bloodstream). But I can't make any statements about the efficacy or safety of any of these supplements, and I definitely wouldn't expect any of the supplements to be effective by themselves, without conventional prescription medications, in the treatment of depression or anything else. I'll reiterate the extreme importance of discussing these details and concepts with your doctor.

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.

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.

General Pharmacological Considerations

This article [Horter and Dressman, 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11259834)] is really interesting, and the authors noted, on the last two pages of text, that the gastric luminal fluid volume can be only 20-30 mL in the fasted state (meaning that the person hasn't ingested anything for 12+ hours, though 14.5 hrs may be required for the stomach to completely empty) and that the USP procedures for evaluating tablet dissolution had been based, at the time the authors wrote the article, in 2001, on nonphysiological pH values (7.5 is not a pH value that's likely to be reached in the GI tract in many people) and surfactant concentrations. The authors also discussed the fact that the rate of dissolution is frequently the most important factor determining the rate of absorption (and, hence, the bioavailability, in many cases). The issue of the luminal fluid volume can be important in determining the rate of dissolution, and the authors noted, for example, that increases in the viscosity of the intestinal luminal fluid, such as in response to food intake, can slow the rate of dissolution and, hence, the rate of absorption. In general, if one wants to maximize the bioavailability of a physiological substance, one should take it on an empty stomach. One might want to spread the total daily dosage out across the day, but it's worthwhile to keep these types of things in mind.

Maximizing bioavailability is not likely to be very important for many supplements, especially if they're in capsule form, etc. For example, I don't think there's any need to try to maximize the bioavailability of encapsulated creatine monohydrate, given that a slight increase in bioavailability is not going to be very important. But, in the case of purine (and pyrimidine) nucleotides, for example, the half-life is extremely short, and the elevation of the concentration of the nucleotide or its metabolites (i.e. other purines) in the systemic circulation is extremely brief, following oral administration. There can be drastic changes in the bioavailabilities of nucleotides, in response to small changes in the rate of dissolution and absorption. Small changes in those parameters have the potential to produce large changes in the bioavailability.

In a related vein, there are still many reports, from articles in the literature and from other sources, of problems with the dissolution of supplements provided in tablet form. It's still a significant problem in the supplement industry, in my opinion. In this context, the issue is not just bioavailability but absorption. If a tablet doesn't dissolve, the absorption and bioavailability will be zero. Consumerlab.com has shown that some tablets essentially don't dissolve at all, and they suggest this complex method for telling if a tablet is going to dissolve (http://www.consumerlab.com/results/hometest.asp). I don't think that's necessary. If a tablet is going to dissolve properly, in my opinion, it should dissolve in a small glass of water in a few minutes. When tablets truly meet dissolution standards, they dissolve in a minute or less. Several years ago, I looked at a lot of reports from Consumerlab.com. They reported that some tablets couldn't be broken with a hammer, and I remember testing some tablets (by putting them in a glass of water) and finding that some of them required 2-3 hours to dissolve. That's obviously not acceptable. I think Consumerlab.com still has some free reports, but I'm not sure. I haven't looked at the site for a few years. A lot of tablets dissolve perfectly, but it's worthwhile to just put a tablet or softgel in a glass of tap water, in my opinion (if one is planning on taking the tablet). I'm saying that that's a way to test if one "sample" tablet of that particular product, from a particular manufacturer, is going to meet some rudimentary dissolution test. If it does, for example, then there's no need to think about it again. In general, though, the dissolution of capsules tends to be more reliable, in my opinion, than the dissolution of those other dosage forms.

Interactions in the Metabolism of Creatine, Adenosine, and Inorganic Phosphate in Acidosis vs. Alkalosis

In this article, Levine et al. (1992) [Levine et al., 1992: (http://radiology.rsnajnls.org/cgi/reprint/185/2/537.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/1410369)] used magnetic resonance spectroscopy (MRS) scans to look at the "phosphocreatine index," which is [PCr]/([PCr] + [Pi]), such that Pi = inorganic phosphate, as a way of quantifying the energy states of cells in different parts of the brain. Levine et al. (1992) also calculated the pH in the brain, by comparing measurements of Pi and PCr, and found a number of changes in the correlations between different pairs of indices in people who had recently had strokes. The authors noted that acidosis generally decreases the PCr/Pi ratio (Levine et al., 1992), and they found that, during the acidotic conditions of ischemia, in the brains of people who had recently (within days or weeks, depending on the patient) had strokes, the pH correlated directly with the phosphocreatine index and with the PCr content (as indicated by the magnitude of the MRS signal) and inversely with Pi. I'm not quite sure how to interpret a change in the intracellular inorganic phosphate levels, but I guess that makes sense that the pH would decrease as the Pi increases. The intracellular (and extracellular, to some extent) Pi levels are thought to generally increase in parallel with the hypoxia or ischemia-associated increases in intracellular AMP levels [Gorman et al., 1997: (http://ajpheart.physiology.org/cgi/reprint/272/2/H913.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/9124455)], and Gorman et al. (1997) also found that a "steady-state," or prehypoxic, depletion of the intracellular phosphocreatine contents, in the hearts of guinea pigs, caused the hypoxia-induced release of Pi and adenosine to decrease. Gorman et al. (1997) also noted that Pi stimulates glycolytic activity, and there's a considerable amount of research showing that the depletion of intracellular levels of either Pi or organic phosphates causes fairly severe deficits in glycolytic and oxidative ATP synthesis. Brautbar et al. (1983) [Brautbar et al., 1983: (http://www.ncbi.nlm.nih.gov/pubmed/6620852)] found that, in response to dietary phosphate depletion/restriction in rats, the total intracellular protein contents of creatine kinase (CK, "creatine phosphokinase"), among the rats, correlated directly with the intracellular Pi concentrations, and both the intracellular Pi levels and creatine kinase levels were decreased in response to dietary Pi depletion. The authors also cited research, on the last page, discussing the capacity of Pi to activate glutaminase and phosphofructokinase activities, and the activities of those enzymes are important in relation to energy metabolism, especially during ischemia or hypoxia. The decrease in the CK protein content was drastic (more than 50 percent) (Brautbar et al., 1983) and is really interesting, in part because the authors only found, in response to dietary Pi restriction, nonsignificant reductions in the intracellular adenosine nucleotide levels and inconsistent changes in the PCr levels (Brautbar et al., 1983). The authors apparently equated the CK protein content with CK activity, and I'm not sure that that would always be a valid assumption (the CK protein content might not correlate linearly with the CK activity, because of phosphorylation of CK by AMPK or by effects of pH on CK activity, etc.). But in humans with renal failure, phosphate supplementation increased ATP levels in the skeletal muscle, compared to controls, but did not change serum phosphate/phosphorus levels. I'd bet that Pi depletion produces changes in PCr or in the PCr index in humans as well as in animals.

Lyoo et al. (2003) [Lyoo et al., 2003: (http://www.ncbi.nlm.nih.gov/pubmed/12850248)] found that the levels of Pi in the brains of humans increased by more than 9 percent in response to creatine (Cr) supplementation, but the authors evidently didn't find any differences in pH between the control and Cr-supplemented groups. Lyoo et al. (2003) also found decreases in the beta-nucleotide triphosphate (beta-NTP) levels (a reflection of ATP levels) and marginal increases in PCr levels, and that trend toward an increase in the PCr/ATP ratio is generally consistent with the increases in PCr/NTP ratio that have been found, in the brains of humans, in response to triacetyluridine or SAM-e administration (http://hardcorephysiologyfun.blogspot.com/2009/06/deoxyribonucleotides-in-mtdna-depletion.html). Lyoo et al. (2003) and others have explained that change in terms of a mass action effect (it's not really ATP depletion and is thought to be evidence of an increase, not a decrease, in intracellular high-energy phosphate levels) that would operate in the overall equilibrium of the intramitochondrial CK enzymatic reaction (the more important CK equilibrium, as I recall) (it's noteworthy that the intramitochondrial ADP pool can be somewhat independent of the cytosolic ADP pool, however):

Cr + ATP <---> PCr + ADP + H(+)

I think that ATP disodium is much more potent than SAM-e and would require lower dosages (expressed both in terms of mg of adenosine that can be derived from ATP disodium, in comparison to mg adenosine that can be derived from SAM-e, and in terms of the dosages of ATPNa2 and SAM-e) to produce effects. Low-dose creatine (1-3 grams/day) has potential, in my opinion, to work in concert with ATPNa2 and uridine or triacetyluridine or cytidine (pyrimidines), etc. Those are just my opinions. Pyrimidines have repeatedly been shown to increase the retention of intracellular purine nucleotides during hypoxia or other metabolic insults. I was going to discuss the research showing that CK activity, induced by cellular creatine depletion, generally appears [O'Gorman et al., 1996: (http://www.ncbi.nlm.nih.gov/pubmed/8816948)] or has been shown to [Dzeja et al., 1996: (http://www.ncbi.nlm.nih.gov/pubmed/8662747)] vary inversely with adenosine kinase activity in different cell types. That effect could be partially due to changes in inorganic phosphate availability, but the relationships between the intracellular Pi levels and adenosine kinase (AK) and creatine kinase activities are likely to be complex. Some research has shown that Pi produces allosteric activation of AK (http://hardcorephysiologyfun.blogspot.com/2009/07/these-are-some-articles-showing-that.html), but Gorman et al. (1997) found that Pi inhibited the activity of AK that had been taken from the hearts of guinea pigs. I don't have time to go into the potential mechanisms, but one has to take into account the adenylate charge and the activating effects that AMP (or an increase in the AMP/ATP ratio) and Pi can have on glycolytic enzymes, etc. It's interesting that Gorman et al. (1997) discussed an index that I've almost never seen researchers use. They referred to the cytosolic phosphorylation potential as being an index, or ratio, that correlates (positively) with the energy state of the cell [similar to the adenylate charge, or {[ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP]), with [ ]'s referring to the intracellular molarities of the nucleotides}. The cytosolic phosphorylation potential is (log [ATP]/[ADP][Pi]). That's sort of similar to the phosphocreatine index, but it seems that one might expect the phosphocreatine index to vary almost inversely with the cytosolic phosphorylation potential.

Interactions of Phosphate Metabolism With Energy Metabolism and Adenosine Metabolism

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

Open Discussion on Multiple Sclerosis, UVB, and Energy Metabolism

I was going to mention that one way of looking at the age-dependence of the protective effect of "migration" in multiple sclerosis (migration to an equatorial latitude) is that the axonal transport capacity or generalized "robustness" is greater in people who are relatively "younger" than in people who are older. There's also a latitude gradient in Epstein-Barr Virus-associated malignancies (Burkitt's lymphoma, etc.), but it's not clear if that's explainable in terms of the lesser extents to which people living in Subsaharan Africa have access to health care and nutrition, etc. (http://scholar.google.com/scholar?hl=en&q=Burkitt%27s+lymphoma+latitude). That's not a great search. But the point is that that could provide indirect support to the ideas people have had about late EBV infection being associated with abnormal neural development in the offspring of people who have had late EBV infections (see past postings and the articles cited in them). I just don't think one can ignore that epidemiology (the latitude gradient and research on migration) in relation to multiple sclerosis, and I don't think it's written in stone that there's no capacity for protection after age 15 or something (no capacity for protection to occur in response to migration or to the environmental, protective factors(s) that have been associated with migration to equatorial latitudes). I've seen research suggesting, for example, that migration may be protective through age 27 or something. That would tend to imply that migration *could* be protective at later ages but that people's behaviors, related to time spent outdoors or to nutritional factors that would interact with that or to activity levels and all other things, have become ingrained, perhaps, by the time people are 16 or 17. On average, how many people radically change everything about their habits after age 18? I'm talking about drastic changes in UVB exposure, etc. I mean, I'm sorry to say it (and I'm in no way suggesting that people go out in the sun without talking with their doctor), but 10 minutes of sun exposure at noon, on the hands and face, is not likely to increase serum 25-hydroxyvitamin D levels all that much and is not going to have the kinds of immunomodulatory effects that depend on hundreds of billions of neutrophils infiltrating the UVB-irradiated skin and the immunosuppressive cytokine milieu associated with it. In my past comments, the main thing I wanted to convey is that I don't think people should expect magic from UVB exposure and that there are many other factors that come into play in the etiology of multiple sclerosis. But, for example, Epstein-Barr Virus infects keratinocytes (http://scholar.google.com/scholar?hl=en&q=Epstein-Barr+keratinocytes), and I don't have to say what that means. It means that there's the *potential* for the induction of tolerance to EBV latent and lytic cycle proteins on a kind of mass scale, following UVB exposure. But there's also the potential for that tolerance to turn into seriously aberrant, Th2-driven immunity and to worsen matters. It's difficult to control or predict the responses, and that's especially true in disease states (in which there's potential for disastrous effects). But, then again, UVB is known to suppress both Th2 and Th1 immunity (the Th1/Th2 dichotomy is a bit outdated but still has some usefulness as a crude framework for looking at these things). But my point is that the release of Th1 cytokines almost disappears, in some cases, from the lymph nodes of UVB-irradiated animals, and the idea that everything boils down to vitamin D and hands-and-face, anemic, Victorian-Era, parasol-carrying approaches is a bit absurd to me. That said, I can't make any recommendations on these things, because I can't give medical advice and, to say the least, can't make any guarantees whatsoever about safety. Avis et al. (1995) (http://scholar.google.com/scholar?hl=en&q=sudden+death+sun+exposure+%22multiple+sclerosis%22) discussed case(s) of people with multiple sclerosis dying after sitting in the sun.

I think it's telling that thermoregulatory dysfunction features prominently in multiple sclerosis, but I don't claim to know how it relates to the supposed UVB-mediated trigeminohypothalamic thermoregulation that may occur in humans. Part of the difficulty is that, for example, the neuropathological effects of Epstein-Barr Virus, in some extreme case studies, can be highly diffuse, and that could be explained in any number of ways (in terms of "diffuse" B-cell infiltration or latent infection of astrocytes and microglia and other perivascular, monocyte-macrophage-lineage cells). I tend to think it's a result of astrocytic infection by EBV, but that's just my opinion. The relapsing-remitting quality could be explained in terms of the devastating effects that pro-inflammatory cytokines can have on energy metabolism, and that could explain the apparent absence of overt inflammation in some research in multiple sclerosis (I can't say anything more specific without looking at the specific articles that people have cited, and I don't want to do that now).

I just don't understand why there would be such resistance to the consideration of all the mechanisms at work. I've seen articles make statements that there's no problem with energy metabolism in the brains of people who have multiple sclerosis. That makes no sense, in my opinion, because axonal degeneration implies profound problems with energy metabolism. And if one buys into the idea that a lot of pro-inflammatory cytokines are being released from activated T-cells infiltrating the CNS, then one would expect major problems with energy metabolism from that. Here's a not-very-good search that shows some of the vast amounts of research showing rapidly-induced mitochondrial dysfunction induced by TNF-alpha and other pro-inflammatory cytokines (http://scholar.google.com/scholar?hl=en&q=TNF+mitochondrial+dysfunction+astrocytes). There's one article, there, in which the authors probably discuss the concept that, in my opinion, optic neuropathies are frequently associated with mitochondrial dysfunction and can be caused by that. The energetic demands of neurotransmission in the optic nerve fibers are enormous. I'm not saying that reducing pro-inflammatory cytokine production by T-cells or other cell types doesn't have the potential to improve energy metabolism. I just think that it would help to acknowledge the deficits in energy metabolism that are very likely to exist in a neurodegenerative disease, such as multiple sclerosis, and to try to develop therapeutic strategies for addressing those deficits in more direct ways. These are just my crude, unrefined thoughts on some of these topics, and I'd strongly urge anyone to discuss things with one's doctor before doing anything.

I'm deliberately discussing some of these things from an idealistic, somewhat impractical point of view, because dogged pragmatism and dogma haven't seemed to be all that beneficial. Even something like parenteral guanosine could be viewed as an energy-metabolism-based strategy, because de novo purine biosynthesis is metabolically costly (and the brain has very little capacity for de novo purine biosynthesis). Also, anticonvulsant medications and adenosine receptor activation or modulation are known to be able to increase or preserve the phosphocreatine to creatine ratio and the adenylate charge. Additionally, purine nucleotide availability is likely, in my opinion, to be a limiting or nearly-limiting factor in mtDNA replication and in other aspects of mitochondrial functioning. I'm not saying guanosine would be a cure-all, but I'm just saying that there are many ways to address energy metabolism.

Elevating uric acid (UA) levels intracellularly, in neurons and astrocytes, such as through the administration of exogenous, parenteral purine nucleotides, would, in my opinion, have the potential to be therapeutic as a result of, among other mechanisms, the UA-mediated improvements in mitochondrial functioning (as a result of peroxynitrite scavenging). That article I cited awhile back, on UA in relation to the sympathetic nervous system and goal-oriented behavior, found intramitochondrial UA levels of 60 uM or something. That's very significant, in my opinion. The peroxynitrite scavenging effects of UA may not look all that special or unique in some articles, but one has to consider the fact that the suppression of nitric oxide (NO) output from activated macrophages, by UA, can occur in the face of these massive increases in the output of iNOS-derived NO. I forget what the variable was--NO output or NADPH oxidase activity--that increases the most dramatically. I think the mRNA or protein content of iNOS can increase 20-50 fold or something, in activated macrophages, and I think the NO output can increase by something like 1000-fold or even more. Some articles show these little graphs of the effects of UA (suppression of NO output by cultured monocyte-macrophage-lineage cells, etc.), and the graphs don't capture what's going on. The UA can suppress NO output drastically at physiological concentrations (of UA), and that's no small feat. NO and peroxynitrite produce strongly detrimental effects on mitochondrial function [a lot of these articles show, rather incidentally or in a manner that doesn't showcase the effects of UA, that UA can ameliorate peroxynitrite-induced mitochondrial dysfunction in various cell types: (http://scholar.google.com/scholar?hl=en&q=mitochondrial+peroxynitrite+urate+OR+uric)].

Adenine Nucleotide Translocase and Mitochondrial Creatine Kinase Activities in the "Maintenance" of mtDNA Integrity; Relevance to Cytotoxic Therapies

This article [Palmieri et al., 2005: (http://hmg.oxfordjournals.org/cgi/content/full/14/20/3079)(http://www.ncbi.nlm.nih.gov/pubmed/16155110?dopt=Abstract)] is really interesting, and the authors described a patient who had no functional activity of the ANT1 isoform of the adenosine/adenine nucleotide translocase protein, which transports ADP into mitochondria and ATP out of the mitochondrial matrix, and had mitochondrial DNA (mtDNA) deletions in the skeletal muscles (and probably also the heart). The ANT1 isoform is the predominant isoform expressed in cardiac and skeletal muscles, and the authors describe the way the person had exercise intolerance and very gradual deterioration in fitness but did not demonstrate decompensated heart failure (Palmieri et al., 2005). The ANT1 isoform is also expressed in the brain, the authors say, but the authors say that the expression of the other isoforms is thought to be significant enough to compensate for the loss of functional ANT1 in different cell types in the brain (Palmieri et al., 2005).

That research may help explain the capacity of creatine supplementation to treat people who have mitochondrial disorders, and the mechanisms by which creatine can sometimes be beneficial in those conditions are not well-understood (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=creatine+monohydrate+mitochondrial). Not all of those studies have shown benefits, however. But creatine has sometimes been shown to decrease or abolish paracrystalline inclusions and other types of structural abnormalities in skeletal muscle mitochondria (or in the myocytes, more broadly). Even though creatine is clearly not a magic bullet, in any way, in the treatment of mitochondrial disorders, in my opinion, I do think there could be some potential, at low dosages, for creatine to prevent the accumulation of mtDNA deletions by its capacity to increase ADP recycling, via the functional coupling of mitochondrial creatine kinase activity (which is in the intermembrane space and intercristae space, outside the mitochondrial matrix) [Dolder et al., 2003: (http://www.jbc.org/cgi/content/full/M208705200)(http://www.ncbi.nlm.nih.gov/pubmed/12621025)] (it's actually thought to form complexes with ANT proteins) to adenine nucleotide translocase activity [Barbour et al., 1984: (http://www.jbc.org/cgi/reprint/259/13/8246)(http://www.ncbi.nlm.nih.gov/pubmed/6330105?dopt=Abstract); (http://scholar.google.com/scholar?q=%22nucleotide+translocase%22+%22creatine+kinase%22&hl=en&lr=)]. Essentially, ANT exports ATP from the mitochondrial matrix, and the activity of mitochondrial creatine kinase ensures, in the presence of an adequate pool of creatine, that ADP is recycled (it's the kinetics of the coupling reactions and spatial proximity, I think, that allow this to occur and to limit the loss of ADP, by diffusion, from the intermembrane space to the cytosol) back into the mitochondrial matrix. That's an oversimplification, but it conveys the concept. The intramitochondrial ADP pool has repeatedly been shown to be maintained, in a creatine-sensitive manner, somewhat or even largely independently of the cytosolic ADP pool, and the effect of creatine can be especially significant during ischemia, etc. This occurs despite the fact that there is no meaningful physical barrier (nothing like the inner mitochondrial membrane) to the diffusion of ADP from the intermembrane space to the cytosol proper. I guess the diffusion can be restricted to some extent, by the outer mitochondrial membrane, but I think the effect of the membrane is not all that significant.

It's important to note, though, that I think the therapeutic dosage range of creatine is fairly small (maybe 1-3 grams/day), but that's just my opinion. I think it has the potential, at higher dosages (or, conceivably, at any dosage in someone with liver disease, for example) to interfere with the transport of other guanidino compounds, including urea cycle intermediates, but that's just my opinion. I've discussed those issues in past postings.

There's actually more research showing that some bisphosphonates can induce apoptosis by forming either xenobiotic-and nucleotide-containing polyphosphates or can inhibit mevalonate-pathway enzymes and increase the formation of endogenous dinucleotide triphosphates, such as inosine triphosphoadenosine (IpppA), and those dinucleotides or dinucleotide-mimetics can inhibit adenine nucleotide translocase activity and thereby contribute to the pro-apoptotic effects of some bisphosphonates in cultured cells, etc. [Monkkonen et al., 2006: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1616989)(http://www.ncbi.nlm.nih.gov/pubmed/16402039)]. This is not always "bad," providing one can effectively target the cells one wants to target. In any case, I have to say that that mechanism doesn't sound very good to me, for many reasons, but that's just my opinion. And a person would obviously want to weigh the risks vs. the benefits in all of these cases and discuss all of those issues with one's doctor before doing anything. There are some case studies included in this search result list that are disturbing to me, and I really shouldn't get into some of these obscure topics sometimes (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=bisphosphonate+mitochondrial+translocase). I end up just finding more and more things I'd almost rather not know about, but I'm probably too idealistic.

Endogenous Formation of Dinucleotide Polyphosphates: Crude Discussion of Interactions with the Mevalonate Pathway, Nucleotide Metabolism, and aa-tRNAs

This is an interesting article [Jankowski et al., 2009: (http://www.brjpharmacol.org/view/0/earlyView.html)], and the authors discuss the fact that dinucleotide polyphosphates (DNPP) are made endogenously and stored in platelets, adrenal chromaffin cells, and also neurons in the brain. The general formula for a DNPP is N-p(x)-N, where N = adenosine, guanosine, uridine, etc., and x = 2 to 7 phosphates linking two 5'-carbons of the ribose moieties of the nucleotides. Some common examples are diadenosine tetraphosphate (Ap4A) and Ap5A, Up4A (U = uridine), etc. That article doesn't discuss that much about their biosynthesis, but it turns out they're formed by aminoacyl-tRNA synthetases, just as the isoprenoid-based nucleotide polyphosphates are (see recent posting). They can also be formed by Ap4A phosphorylases, luciferase enzymes, and guanylyltransferases [Schluter et al., 1998: (http://www.pubmedcentral.nih.gov/picrender.fcgi?doi=10.1172/JCI119882&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/9449703)]. The different DNPP's have agonist and antagonist effects, at low concentrations, on purinergic receptors, and the article by Jankowski et al. (2009) catalogues a lot of those effects on platelet aggregation, etc. They're apparently degraded fairly slowly by various hydrolase enzymes.

So I guess to understand the interactions of cholesterol and nucleotide metabolism, one should read about the regulation of aminoacyl-tRNA synthetases. That's bizarre, but it seems interesting. It looks like the DNPP's have both "good" and "bad" effects, as one might expect, and it sounds like the concentrations can be fairly high, in the millimolar range, in vesicles in neurons or in platelet secretory granules. They basically are thought to act locally, the way purines and other nucleotides act on purinergic receptors. The plasma concentrations of different diadenosine oligophosphates/polyphosphates are 0.18 to 0.89 uM, but Jankowski et al. (2009) think that they may act locally on platelets and influence platelet aggregation under some circumstances. It sounds like one effect might be on phosphate homeostasis, as the authors of that article on isopentenyl-ATP derivatives were implying and suggesting. It seems like there may be a tendency to focus a lot on the effects of DNPP's on purinergic receptors, but it sounds like the intracellular effects might be more important. I haven't read much of anything on this topic yet, and there's probably some research about that type of thing. Maybe they regulate cholesterol metabolism or interfere with nucleotide transport. Some genetic mutations that affect nucleotide transport can cause mtDNA depletion, and then there are the more short-term effects (of DNPP's), such as on respiration, that could potentially occur via the inhibition of the adenine nucleotide translocase transporter. I wonder if there's anything on mRNA stability or something like that or on transcription. Presumably there are all sorts of potential pathological effects, but I actually don't know anything about the mechanisms governing the intracellular transport of DNPP's. I also don't know what factors regulate their formation by the aminoacyl-tRNA synthetases or by other enzymes. Schluter et al. (1998) say that the half lives of various DNPP's range from 49 to 69 minutes, and those are much longer than the half-lives of purines. The half-life of plasma adenosine is about 0.5 to 1.5 seconds.

It's interesting that Schluter et al. (1998) say that the aminoacyl-tRNA synthetases transfer the AMP of an aminoacyl-AMP to a nucleotide diphosphate or triphosphate and form the DNPP and also release an amino acid. I wonder if there's some kind of specificity to the aminoacyl-tRNAs that form specific DNPP's. If there were (I have no idea if there is), then that could explain some of these strange effects of different amino acids. That could explain some of the puzzling effects of glutamine, such as its antiapoptotic effects. For example, glutaminyl-tRNA synthetase [Ko et al., 2001: (http://www.jbc.org/cgi/content/full/276/8/6030)(http://www.ncbi.nlm.nih.gov/pubmed/11096076?dopt=Abstract)] produces antiapoptotic effects, in a glutamine-dependent manner, by obscure mechanisms. Ko et al. (2001) discuss some of the mechanisms by which glutaminyl-tRNA synthetase may contribute to the supposed antiapoptotic effects of glutamine. Maybe there's some intermediary influence of glutaminyl-tRNA-derived DNPP's in the antiapoptotic effects of glutamine, under some circumstances. I mean, maybe the DNPP formation is facilitated in some way by protein-protein interactions of glutaminyl-tRNA synthetase and some other protein [even the apoptosis signal-regulating kinase 1, discussed by Ko et al. (2001), that interacts with glutaminyl-tRNA synthetase, etc.]. Those are crude ideas, but it's interesting. The article by Ko et al. (2001) discusses the effects of glutamine on various mitogen-activated protein kinase pathways.

Formation of ATP Derivatives of Intermediates in Cholesterol Biosynthesis: Potential Relevance to Energy Metabolism in the Brain, etc.

This article [Sillero et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19414000)] discuss the capacity of some bisphosphonates to inhibit farnesyl pyrophosphate synthetase, thereby inhibiting cholesterol biosynthesis and also the posttranslational prenylation of GTPases and other proteins. The authors also found that a variety of ligase enzymes, such as DNA ligases, apparently, cleave ATP and, as part of their catalytic mechanisms, attach the AMP derived from that cleavage to the ligase enzyme or to a cosubstrate, X (it's not clear what the cosubstrates are, and I don't feel like looking them up). This forms an E-X-AMP or E-AMP complex of AMP with the enzyme. Some of the ligase enzymes can then transfer the AMP to isopentenyl pyrophosphate (Iso-pp) or to a bisphosphonate and form Iso-pppAdenosine or "ATP derivatives" of bisphosphonates that may contribute to the induction of apoptosis in osteoclasts or monocyte-macrophage lineage cells, including osteoclast progenitor cells. One mechanism by which these ATP derivatives are thought to cause apoptosis is through the inhibition of the mitochondrial adenine nucleotide translocase transporter. Faust et al. (1980) [Faust et al., 1980: (http://www.jbc.org/cgi/reprint/255/14/6546.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/7391033)] found that aminoacyl-tRNA synthetases could form delta2-isopentenyl-tRNA, and those tRNAs are similar to the ATP derivatives, discussed above, in the sense that the isopentenyl group is transfered to the N6 nitrogen atom of an adenosine that is part of the tRNA. Faust et al. (1980) claim that, when cholesterol levels are low in some cells, more mevalonate is diverted into cholesterol formation than into ubiquinone and isopentenyl derivatives (isopentenyladenosine is one commonly-discussed derivative, and I'm actually forgetting if that's the same thing as Iso-pppAdenosine or is different from that). I don't know that that's true, because supposedly the ubiquinone pathway, at least, is maintained very efficiently under almost all circumstances, as I recall. It's only when HMG-CoA reductase activity has become greatly decreased that ubiquinone formation decreases, as I remember. But Faust et al. (1980) nonetheless show that inhibiting HMG-CoA reductase can decrease mevalonate levels, as is well-known, and thereby reduce the formation of some of these endogenous isopentenyl-adenosine derivatives (either free Iso- derived species or tRNA-bound derivatives, etc.).

This could be relevant to the roles that decreases in the neuronal or astrocytic cholesterol contents may play in the etiologies of neurodegenerative or psychiatric conditions, in my view. If the cellular cholesterol concentration that is capable of influencing HMG-CoA reductase activity in a cell in the brain were to be too low to maintain low levels of these nucleotide derivatives of isoprenoids, it's conceivable that that could, in some way, influence energy metabolism. The isopentenyl adenosine derivatives might interfere with nucleotide transport or contribute to DNA damage, etc. I haven't thought about the details of this, but Sillero et al. (2009) discuss the very large capacity of the mevalonate-derived isoprenoids and other cholesterol-biosynthetic intermediates to sequester pyrophosphate, and that could compromise adenosine nucleotide salvage. Fructose, for example, can cause ATP depletion in the liver by sequestering phosphate in one of the fructose bisphosphates (I forget which one), etc. These are fairly crude suggestions, but it's an interesting area of research.

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

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

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

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

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