Glutamine and Uridine in Hexosamine Biosynthesis and Glycosylation Reactions

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

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

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.

Fractionation of Fixed-Dosage Preparations of Uridine or Triacetyluridine

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

More Data on the Relative Bioavailabilities of Uridine and Triacetyluridine

These articles [Al Safarjalani et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/15729584); Ashour et al., 1996: (http://www.ncbi.nlm.nih.gov/pubmed/8687475)] show that the relative bioavailability of oral uridine (URD) at a dose of 1320 mg/kg, in comparison to the response to intraperitoneal URD, was about 7.7 percent (Ashour et al., 1996). Al Safarjalani et al. (2005) looked at the relative bioavailability of URD at different dosages, and I don't have time to check the full text of that article right now. Ashour et al. (1996) found that the relative bioavailability of triacetyluridine (TAU) (oral vs. parenteral) was 53 percent. The 7.7-percent value is about ten times the relative bioavailability, crudely calculated, in that older study I linked to in a recent posting, and that difference might be attributed to differences in methodologies or to the limits in oral bioavailability that one would expect to see, because of the low solubility of uridine, at higher single dosages of uridine (it would slow the entry into solution and essentially mimick the effects of a time release preparation, because the absorption of uridine would gradually allow more uridine to enter solution in the intraluminal fluid in the small intestine, etc.).

Solubility and Bioavailability Issues With Uridine, etc.

I was going to mention that the relative bioavailability of free uridine is almost certainly not as low as that article (the one I cited in the last posting) seems to suggest. Uridine is insoluble at concentrations above 10 mM, and I had to find some obscure article on plants or something to find any data on its solubility. I know the solubility of uridine monophosphate disodium is substantially higher (and so is the solubility of uridine diphosphoglucose), but I can't find quantitative solubility data on those uridine derivatives. The way you can tell is that the osmotic GI effects reported in the literature are a result of low solubility, almost certainly. The absence of reports of those issues from human studies using oral uridine diphosphoglucose or the uridine monophosphate disodium strongly implies, as other authors have intimated, that they are substantially more soluble. There's no quantitative solubility data on uridine salts in the Merck Index or online. But there's obviously no solubility issue with triacetyluridine, to any significant degree, in my opinion. But that issue would skew data on relative bioavailability, above a certain concentration, because there would be a plateau of bioavailability of oral uridine, past a certain dosage. Also, some of these ridiculous reports of strange reactions, in rodents, to i.v. uridine are, in my opinion, obviously explainable either in terms of the vehicles used in i.v. preparations (there are countless examples of this in the literature) or in terms of the precipitation of some of the uridine, potentially. Maybe if they'd actually collected solubility data, they wouldn't have botched that sort of thing.

Think about the fact that there's no quantiative solubility data on uridine salts. I looked online for maybe 45 minutes. I could not find any. That's pathetic. It's shocking and shameful, with all of this big public-relations "stuff" about sequencing the human genome and about genes and gene expression and all of that. It's just one example of the deep, deep problems in a lot of these areas. Uridine and its prodrugs are not cure-all's or anything and are only useful up to a point, but think of the wasted money and time in relation to the subjects I've discussed in the last postings. They could have provided much more help than they did to all of those people with brain injuries and degeneration, just by actually exploring the relationship between the therapeutic effects and the increases in plasma uridine (from CDP-choline or a soluble uridine salt or a uridine pro"drug"). It's like hearing about someone living in a big, lavish mansion who's passed out on i.v. opiates or something, all the time, drunk on the hopes of big interventions that never seem to materialize or live up to the empty promises and hype, and who can't care for his or her kids, who are sitting in the corner starving to death (while there are servants running all over the place and tending to the passed-out person's every need). I know these are harsh statements, but this is just disgusting. Every time I start writing or talking about it, I can hardly contain my disgust.

Paralysis in Research on Pyrimidines

There isn't even an easy way to express the comparison, despite the relative simplicity of the issue. But the animal studies and cell culture studies have generally shown that, to produce robust neuroprotection against damage due to mitochondrial toxins (i.e. 3-nitropropionic acid), the plasma uridine level should be kept above 80 uM for at least half the day (12+ hours). It's similar in cell culture studies. The extracellular uridine concentration has to be in that vicinity of 100 uM or so, ideally. That doesn't mean that there wouldn't be beneficial effects at lower plasma levels, in the context of less severe mitochondrial impairment. The assumption has generally been that that "requirement" stems from the need to supply pyrimidines to polymerase-gamma (based on the whole rho-naught, mtDNA-depleted cells that display uridine and pyruvate auxotrophy), to maintain mitochondrial DNA replication, but it's fairly clear, based on a number of animal studies, that that's not likely to be an especially important mechanism in vivo. I should say that the supply of the pyrimidines themselves (per se), for mtDNA replication, is, at least, unlikely to be a major mechanism. It's conceivable that the maintenance of the astrocytic glycogen content, for example, and glycogen utilization and glycolytic ATP production (as has been shown in multiple organs, including the skeletal muscles, heart, etc.) is required for the maintenance of mtDNA replication. But I think it's basically that it buffers energy metabolism by various mechanisms, such as by increasing glucose uptake. That article that I discussed recently, on cultured deoxyguanosine-kinase-deficient cells, basically shows that ATP depletion and guanosine nucleotide depletion can compromise mtDNA replication, but that's not really anything new. Additionally, there's a considerable amount of research showing that CDP-choline can increase glucose uptake, and it's probably just a result of the same types of mechanisms by which uridine exerts its effects.

But as far as the issue of choline goes, how could choline *possibly* produce any protective effect? There's already going to be a gross excess of free choline during ischemia, and there just isn't any mechanism by which it could produce protection. It's like expecting magic, expecting growth in cells without supplying any glucose or any alternative substrates to prevent the cells from dying. Why would one expect that? There's just no conceivable mechanism by which dumping extra choline into the brain could contribute to neuroprotection, and there's just this endless, bizarre, ongoing problem with all the junk animal research on choline.

For example, if I were trying to get my candidate elected, in a national election, would sending out "magic" vote-getters, to try to get "special votes" that would carry extra weight, bypass the will of the voters or make 1+1 = 270 instead of 2 electoral votes? That's a terribly obnoxious analogy, but that's what some people are really talking about with some of these approaches to neuroprotection. There just isn't any way around the issue of energy metabolism, and there never will be.

But it's like there's no one who's interested in mechanisms anymore, except the business about maintaining phospholipid metabolism. It's fine to focus on that, but why does uridine or cytidine decrease free fatty acids (FFA's) during ischemia? Is it just the one enzyme that's affected (i.e. CTP:phosphocholine cytidylyltransferase)? Is that the only enzyme or protein whose activity or functions is/are going to be affected by pyrimidines? I think it just boils down to ATP-buffering, and that doesn't mean there shouldn't be any more research or that it's a magic bullet. I guess it wouldn't even matter all that much what the mechanisms are, if something like uridine were actually being made use of. A lot of the research on uridine in humans should have been done 50 years ago. There's research that's 30 or 40 years old, showing that uridine can protect cultured cells against glucose deprivation, etc. It's nothing special, necessarily, but uridine makes most of these other things people talk about look like child's play.

It's that aspect of all of this, this area of research, that just feels like something that's been dying for a very long time, slowly and intractably, crying out for some kind of sense. It's this kind of motionless and paralyzing death, while everything seems to be moving more and more rapidly, on the surface, at least, and I just almost can't believe it sometimes.

Note on Triacetyluridine and Uridine Prodrugs

I was going to mention that triacetyluridine, as discussed in a couple of recent postings, is not only being sold on that obscure website. If one searches on "google shopping," for example, one sees it for sale there. Some of the preparations, however, are sold at either 100 or 200 times the price that triacetyluridine is sold for on that obscure website that I linked to in a posting last winter. I forget the precise difference, but I did a quick calculation on it, when I did the posting in the winter. The company that anyone can look up the name of and that had been researching triacetyluridine has apparently moved on to the testing of RG2417, another uridine prodrug. I'm almost certain that it's just some other acyluridine, but the company is literally not revealing the identity of the compound. I can't find it online. The authors of this article [Tochigi et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18068248)] said, in 2008, that "A large-scale clinical trial of uridine (RG2417) for bipolar depression is underway" (Tochigi et al., 2008, p. 189). It's a uridine prodrug and is probably one of those more lipophilic acyluridines. That would be my guess. The description of it as an "intracellular mood stabilizer" (http://www.marketresearch.com/map/prod/1344386.html) implies that it may be more lipophilic than uridine or triacetyluridine and that it enters cells more readily (i.e. because it's more lipophilic, presumably to enhance entry into the brain). That's just my guess, though, my opinion. I'm not sure what an "extracellular mood stabilizer" would be--I guess one that doesn't actually enter cells. Triacetyluridine is or was designated RG2133 and was also named PN401, because a different company had previously owned the patents or been researching it. Maybe the secrecy is because of all of the controversy that occurred when PN401 (triacetyluridine) was first being researched in mitochondrial disorders. The people who had mitochondrial disorders really wanted PN401 and wanted it to be researched more, and there was just a mess with it. I think the people who wanted it knew, on some level, that it was so close to being identical to uridine itself that....well, I shouldn't talk about all of these issues anymore.

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

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

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

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

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

Folate and UVB Papers in Pdf Format

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

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

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

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

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

The Mesolimbic Reward Pathways in Depression

The authors of this article [Nestler and Carlezon, 2006: (http://www3.utsouthwestern.edu/contecenter/refs/Nestler2.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/16566899)] discuss the potentially-important roles that dysregulations of the mesolimbic and mesocortical, dopaminergic pathways may play in the etiology of major depression. Carlezon is a researcher at Harvard Medical School and has contributed to a lot of really interesting research, such as the research showing antidepressant effects of uridine or cytidine in animal models of depression [Carlezon et al., 2002; Carlezon et al., 2005, both cited and discussed here: (http://hardcorephysiologyfun.blogspot.com/2009/02/potential-psychiatric-pitfalls-in.html)]. Nestler and Carlezon (2006) note that the tendency has been to focus on changes in the hippocampus or prefrontal cortex in depression, etc. It's stunning to me that someone had to write an article like this. Of course one would expect, in my opinion, the mesolimbic reward pathways to be dysregulated in people with depression. It's also noteworthy that some hippocampal neurons project to the prefrontal cortex and are thought to be important in the regulation of dopaminergically-mediated working memory performance. For example, Seamans et al. (1998) [Seamans et al., 1998: (http://www.jneurosci.org/cgi/content/full/18/4/1613)(http://www.ncbi.nlm.nih.gov/pubmed/9454866?dopt=Abstract)] discussed the way in which dopaminergic neurons, originating in either the ventral tegmental area (VTA) or nucleus accumbens or other sites in the striatum [Carr and Sesack, 2000, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/03/adenosine-and-guanosine-in-animal.html)], project to and form synapses with GABAergic interneurons in the prefrontal cortex and are thought, via the activation of D1 dopamine receptors on those GABAergic interneurons, to exert a largely inhibitory influence on the burst firing patterns of glutamatergic pyramidal neurons that originate in the prefrontal cortex and provide monosynaptic inputs back to the dopaminergic neurons in the VTA, in the midbrain. The "regulatory" effects of glutamatergic inputs, originating in the prefrontal cortex, to the VTA are important for maintaining the normal burst firing patterns of VTA neurons, and the burst firing patterns of dopaminergic neurons in the VTA are crucially important for processes such as reward-based learning, cognitive functioning, and motivation, etc. Dopaminergic neurons in the VTA can also produce excitatory or inhibitory effects on glutamatergic neurons in the prefrontal cortex by modifying the excitatory or inhibitory effects of hippocampal neurons that provide inputs to the same classes of GABAergic interneurons that regulate the layer V glutamatergic neurons that project back to the VTA. In any case, those are some ways the reward pathways interact with the hippocampus and prefrontal cortex, and many of those interactions would, in my opinion, be relevant to an understanding of dysregulations of the reward pathways in people with major depression.

Elevation of Plasma Hypoxanthine During and After Glycogen-Depleting Exercise: Relevance to Energy Metabolism and Nucleotide Turnover in the Brain

This is a really interesting article [Sahlin et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10502075)], and the authors found that plasma hypoxanthine increased by a mean of 7.78-fold during exercise in humans. There was some variation in the magnitudes of the increases in plasma hypoxanthine among individuals. The increases in plasma uric acid (urate) and xanthine, which is derived from the metabolism of hypoxanthine by xanthine oxidoreductase (xanthine oxidase is technically a modified form of xanthine reductase that's been modified by proteolytic cleavage, etc., but people typically refer to the enzyme activity as being "xanthine oxidase" activity), were larger in terms of the amounts of those purines formed, but the percent increases were lower (mean increases of 5 percent for urate and 223%, or a 2.23-fold increase, for xanthine). The effect of that increase in plasma hypoxanthine on the brain should not be underestimated. In a past posting, I discussed some of the research showing neuroprotective effects of remarkably low doses of hypoxanthine [Mink and Johnston, 2007, cited and discussed here: (http://hardcorephysiologyfun.blogspot.com/2009/03/protection-against-postischemic-damage.html)]. There's still the fact that the exercise-induced increases in neuronal activity in the brain would be expected to substantially, albeit transiently, increase the rates of purine export from neurons. I can't immediately find any articles showing elevated cerebrospinal fluid hypoxanthine and xanthine and urate levels, following exercise, but it's very likely that those elevations would occur, in my opinion, especially following high-intensity exercise. So that supposed depleting effect of exercise on neuronal and astrocyte purine nucleotide levels would be expected to lessen the impact of an increase in the plasma hypoxanthine level. I would think that, as a person's muscle mass increased over time, the muscles' capacity to export hypoxanthine would be increased and would produce more significant effects on the brain. But the person would, in my opinion, keep having to push the limits and produce glycogen depletion, as discussed below. Other researchers have discussed the effects of muscle-derived purines on the brain. There's one article from 1978 or 1979 that discusses the potential effects of exercise-induced elevations in extracellular ATP, released from endothelial cells, on the brain, but the authors focused too much on ATP per se. Extracellular ATP is rapidly degraded to ADP and then adenosine and hypoxanthine, etc., and so one would expect to see much more of an effect of exercise on adenosine or hypoxanthine than on ATP. I mean that the purines that might be expected to enter the brain in significant amounts, during exercise, and to also exert meaningful effects on nucleotide pools in neurons or astrocytes would be hypoxanthine or, conceivably, adenosine and not ATP, in my opinion. Xanthine is not salvaged efficiently (but can, in fact, be salvaged in small amounts to xanthosine and then guanosine), but hypoxanthine and adenosine are salvaged relatively efficiently by cells in the brain.

Sahlin et al. (1999) discuss the fact that researchers have generally found glycogen depletion from the skeletal muscles to be a prerequisite for the most pronounced, exercise-induced increases in plasma purines, including hypoxanthine. True glycogen depletion from a muscle group generally requires exhaustive exercise, and the research has generally shown, in my opinion, that resistance exercise produces more-pronounced degrees of glycogen depletion and purine depletion from the muscles [the general idea is that ATP depletion causes a loss of the capacity to salvage adenosine (and also guanosine), and this causes inosine monophosphate to accumulate and be converted into hypoxanthine and xanthine in the muscle cells] than exercise at low intensity does. Hellsten et al. (1998) [Hellsten et al., 1998: (http://ajpendo.physiology.org/cgi/content/full/274/4/E600)(http://www.ncbi.nlm.nih.gov/pubmed/9575819)] discuss research showing that a high-intensity exercise program causes a 20 percent decrease in the total adenine nucleotide contents of skeletal muscles (when people are not exercising, meaning post-exercise and all the time). That's a remarkable fact and suggests to me that some benefit might be derived from low-dose adenosine or guanosine supplementation, particularly early in an exercise program. But that's my opinion. The effects of hypoxanthine on the brain should not be underestimated, and a single exercise session that produces an 8-fold elevation in plasma hypoxanthine levels, during the hour or few hours following exercise, could have a significant effect on the pools of adenine nucleotides, in particular, in the brain. Mink and Johnston (2007), cited above, discuss the fact that hypoxanthine appeared to be salvaged to a large extent, even during ischemia. The brain has an extremely low capacity to make purines de novo and depends almost entirely on purines exported from either the endothelial cells lining the cerebral blood vessels or from the blood.

Of course, creatine and glutamine have been shown to augment the salvage of purine or pyrimidine nucleotides in various articles [cited and discussed here or in other postings: (http://hardcorephysiologyfun.blogspot.com/2009/02/interactions-of-glutamine-and-arginine.html)], but any supposed improvement in purine or pyrimidine salvage, in the brain, that might occur in response to the administration of those types of supplements might be offset by the increase in, for example, purine nucleotide export that could accompany a creatine- or glutamine-induced increase in exercise intensity. I don't think that's the way it would work, though, as long as the doses of creatine or glutamine are kept low, but that's just my opinion. For example, the combination of glutamine and inosine, which is hypoxanthine riboside (hypoxanthine attached to ribose to make a nucleotide), [Hodges and Snyder, 2004, cited here: (http://hardcorephysiologyfun.blogspot.com/2009/02/contribution-of-glutamine-to-pool-of.html)]. That's just my opinion. In my opinion, low doses of glutamine, creatine and adenosine and guanosine might produce some sort of buffering effect on brain energy metabolism and purine nucleotide pools. Creatine increases or "stimulates" oxidative metabolism, in part by maintaining the intramitochondrial ADP pool, and this would be expected to increase the extent to which glutamine, upon its metabolism into glutamate, can be metabolized into alpha-ketoglutarate and undergo oxidative metabolism in neurons or astrocytes in the brain. People discuss creatine as if it participates only in anaerobic metabolism, but this is just not the case. Creatine can prolong "aerobic" exercise and is known to increase oxidative glucose utilization in cells, etc.

Similarly, cytidine was recently shown to decrease the glutamine+glutamate pool in parts of the brains of humans [Yoon et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19194376)], and that type of effect could be explained in terms of an increase in the flux of substrates through the nonoxidative pentose cycle, via the ribose-1-phosphate derived from cytidine catabolism in the brain, and glycolytic pathways. Uridine is converted into cytidine, and ribose derived from purines might be expected to produce a similar effect, to some extent. Hodges and Snyder (2004), in fact, discuss research showing that either glucose or glutamine can serve as an energy substrate for cultured cells but that inosine or some other "pentose source," meaning ribose derived from inosine, had to be present, in combination with glutamine, to maintain ATP levels in cultured cells that lacked a source of glucose. Exogenous nucleotides have been shown to elevate lactate levels in many articles, and those effects could be partly attributable to the ribose-induced increases in the activities of glycolytic enzymes. The effect of cytidine could also have been the result of some effect on receptors that bind pyrimidine triphosphates, etc., but that type of mechanism, in my opinion, is less plausible. Nonetheless, I don't think ribose, by itself would substitute for uridine, past a certain point.

My overall point is that some of these energy substrates, such as creatine and glutamine and ribose, can increase the flux of intermediates through one pathway and produce beneficial effects that might, in my opinion, be expected to be limited by the "depleting" effect that might result from that shift in the metabolism of one or another intermediates. At high doses, however, these types of supplements start to produce really complicated effects. The glutamine-induced decreases in plasma free fatty acids, for example, could, in my opinion, begin to become counterproductive, with respect to the brain, at higher dosages. Obviously, one would want to discuss any of these supplements with one's doctor before taking any of them or beginning any kind of exercise program.

Adenosine, PKA Activity, CREB Activation, Synapsin I Activation, and the Actions of Antidepressants

These articles [Consogno et al., 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11543736); Zanotti et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9833637)] show that S-adenosylmethionine (SAM-e) increased the activity of calmodulin-dependent protein kinase II (CaMK-II) and protein kinase A [cyclic adenosine monophosphate (cAMP) dependent protein kinase] in various parts of the brain, and some of these increases were similar to changes that are sometimes produced by various antidepressant drugs. I think those effects are due to the actions of adenosine, derived from the exogenous SAM-e, as I've discussed in recent postings (http://hardcorephysiologyfun.blogspot.com/2009/03/adenosine-and-guanosine-in-animal.html). For example, Cheng et al. (2002) found that the activation of A2A adenosine receptors produced an increase in (or normalization of, in the face of the inhibition of one of the intracellular, mitogen-activated protein kinase cascades that is activated by NGF) the NGF-induced neurite growth in cultured neurons, and this effect of A2A adenosine receptor activation was dependent on the PKA-induced activation (phosphorylation) of cAMP response element binding protein (CREB) [Cheng et al., 2002: (http://www.jbc.org/cgi/reprint/277/37/33930)(http://www.ncbi.nlm.nih.gov/pubmed/12114502?dopt=Abstract)]. Similarly, Diogenes et al. (2004) [Diogenes et al., 2004: (http://www.jneurosci.org/cgi/reprint/24/12/2905)(http://www.ncbi.nlm.nih.gov/pubmed/15044529?dopt=Abstract)] found that both A2A adenosine receptor activation and PKA activity were required for the in vitro neurotrophic effects of brain-derived neurotrophic factor (BDNF) on hippocampal neurons. Some of these articles (http://scholar.google.com/scholar?num=100&hl=en&lr=&safe=off&q=CREB+adenosine+activation+A2A+OR+A3) show that adenosine receptor activation can produce an increase in the phosphorylation of CREB by PKA and in various phospho-CREB-induced changes in gene expression in various cell types. An increase in CREB phosphorylation can result from an increase in the activity of CaMK-II, p38 MAPK, PKA, or other intracellular kinase enzymes or signalling pathways, and increases in phospho-CREB levels, in the hippocampus, among other parts of the brain, have traditionally been viewed as one change that accompanies the response to an antidepressant treatment (an antidepressant effect). The reality is probably much more complex, as noted by Manier et al. (2002) [Manier et al., 2002: (http://www.ncbi.nlm.nih.gov/pubmed/11793165)], given that antidepressants may produce their effects, under some conditions or in some people, by decreasing phospho-CREB levels. There's also research showing that guanosine can increase CREB activation or cAMP levels, but I can't get into all of that now. An increase in CREB activation is just one of many changes that can occur in the brain in response to some antidepressants, but that's the type of mechanism that could, in my opinion, conceivably explain the ways in which longer-term antidepressant effects could emerge out of those short-term antidepressant effects of guanosine and adenosine in animal models of depression (http://hardcorephysiologyfun.blogspot.com/2009/03/adenosine-and-guanosine-in-animal.html). I think an increased in phospho-CREB levels couldn't have occurred over those short-term experiments, but an increase in cAMP could have occurred. I tend to think those short-term effects are more inhibitory and have more to do with inhibition of glutamate release by guanosine or adenosine, etc., as implied by the results. The activation of A1 adenosine receptors and other adenosine receptor subtypes can preserve phosphocreatine, etc.

Incidentally, SAM-e, in one of those articles I cited at the beginning of this posting, was also shown to increase synapsin I protein levels (Consogno et al., 2001), and synapsin I is a protein substrate of CaMK-II (synapsin I is phosphorylated by CaMK-II and thereby enters the cytosol) that can regulate neurotransmitter release. SAM-e produced an increase in the cytosolic, "soluble" synapsin I concentration that was, therefore, likely to have been the result of a SAM-e-induced increase in CaMK-II activity. There's also a considerable amount of research showing reductions or changes in synapsin I protein levels in people with depression, etc. (http://scholar.google.com/scholar?q=%22synapsin+I%22+antidepressant&hl=en&lr=).

Interactions of Caffeine With Purine Metabolism, Ribose, and Uric Acid

The authors of this article [Herrick et al., 2009: (http://linkinghub.elsevier.com/retrieve/pii/S0306987709000061)] suggest that people could combine D-ribose with caffeine to augment the effect of caffeine and conceivably decrease the adverse effects associated with caffeine intake. The authors are essentially saying that ribose could augment ATP production and either decrease or increase the export of adenosine and its nucleotides from cells, meaning neurons, that have been stimulated by caffeine, etc. There's some validity to this suggestion, but, in my opinion, using purines or uridine as a source of small amounts of ribose would be a safer and more effective approach in the long term. Inosine monophosphate is ~43 percent ribose, and some similar percent of adenosine and guanosine are ribose. I don't feel like looking up the molar masses. In my opinion, high-dose ribose is not really a good idea, but I suppose one approach would be to combine small doses of ribose with purines and uridine or cytidine (uridine has been shown to elevated the cytidine and uridine nucleotide pools to significant extents, and so one doesn't need to take cytidine, really), etc. Barsotti and Ipata (2002) [Barsotti and Ipata, 2002: (http://www.ncbi.nlm.nih.gov/pubmed/11841784)] note that ribose has been shown to more effectively augment ATP repletion, following ischemia, when purines or purine bases are given along with the ribose (references 2 and 4, p. 130). Other articles have also shown that to be the case [Smolenski, 2000: (http://www.actabp.pl/pdf/4_2000/1171-1178s.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/11996106)].

One major reason I say that ribose from purines would, in my opinion, be safer is that ribose has been shown to increase the export of purine nucleotides and nucleosides from skeletal muscle cells and other cell types (including cells in the liver, etc.). The "purine-wasting" effect is not nearly as large as the effect of xylitol (I've cited much of this research in postings in December and early January) or fructose, but there is the potential for that to occur, in my opinion. That essentially means, in my view, that there would be short-term export of purines that would appear to be beneficial and long-term sequalae that would not be desirable. A similar effect occurs with something like zinc, which shows all these apparent "antidepressant" effects in short-term animal studies. Zinc increases the export of adenosine and other purines, partly by serving as a cofactor for a number of nucleotidase enzymes that degrade intracellular and extracellular nucleotides. That can produce short-term benefits, but the articles showing neurotoxicity from excessive zinc supplementation or from derangements in zinc homeostasis, in the absence of supplementation, are almost endless in numbers and are just absolutely appalling to see. I collected about a hundred of them in a list, and I'll try to link to a large number of them one of these days. They're not pleasant to look for or to read. That's an extreme example, in any case. Also, adenosine is exported from neurons in a generalized manner in response to neuronal activity, and that would be part of the rationale for providing actual exogenous purines instead of ribose. This is basically the same thing I've been saying over and over again, and there are almost innumerable articles showing that purine export is a generalized response to the excitation of neurons, either by electrical stimulation or pharmacological manipulations that increase excitatory neurotransmission. This effect, again, to the extent that it would be therapeutic, might not be long-lived in the absence of some attempt to address the resulting deficit in the intracellular purine pools. I came across a reference to an old article suggesting that antidepressants may exert some of their effects by increasing adenosine availability (this was discussed in the context of the capacity of low levels of adenosine to produce activation, rather than inhibition, of adenylate cyclase) [cited as reference 7 on p. 598 in Cooper et al., 1980: (http://www.ncbi.nlm.nih.gov/pubmed/6162091)]. I'm not sure if that cited article is talking about a reduction in the export of adenosine or about the export of adenosine from astrocytes leading to the import of adenosine into neurons. But I have multiple articles showing that either exogenous guanosine, adenosine, or inosine can elevate cAMP in various cell types, and I don't feel like linking to them right now. cAMP signaling is really complex, though, and an increase in the activities of cAMP-dependent protein kinases can be "pathological" or undesirable under some circumstances, and adenosine exerts a very complex set of effects on cAMP signaling.

Ribose mainly would contribute to the pool of intermediates in the nonoxidative pentose cycle, and this would mainly assist, to some extent, in the salvage of purines and in the provision of ATP by glycolysis, etc. (the activities of the enzymes of the de novo purine biosynthetic pathway are very low in the brain, and any increase in de novo inosine monophosphate formation from exogenous ribose alone would, in my opinion, be fairly minimal).

This suggestion about ribose is actually sort of minimally interesting in the context of the bizarre formulation of some of these well-known energy drinks advertised heavily on tv. I'm not going to say the brand, but I looked up the ingredients to see what was supposed to be so special about one of them. I see nothing very special about it and don't see the appeal of it. But an ingredient that stands out, in combination with caffeine, as being unusual is glucuronolactone. This is metabolized into glucuronic acid, and some of labeled glucuronolactone is converted into L-xylulose and then ribose [Hiatt, 1958: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1062823&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/13575548)]. So, in my opinion, glucuronolactone is like a third-rate substitute for ribose (the conversion of L-xylulose into xylulose-5-phosphate is ATP-consuming, and this ATP consumption and phosphate sequestration is basically the mechanism whereby xylulose produces more purine depletion than ribose, etc.). These types of differences in the point of entry into the pentose cycle have been shown, fairly clearly, in my opinion, to produce surprisingly significant effects on ATP levels and phosphate sequestration, and it's partly because of the large amounts of the sugar(s) entering cells at one time.

Another advantage of providing the actual purines, as a source of ribose, would be, in my opinion (apart from the peroxynitrite scavenging effect of uric acid), the capacity of uric acid (urate) to influence purine metabolism indirectly (also, oral inosine has been shown to elevate plasma hypoxanthine and xanthine in humans, and the contribution of those elevations in hypoxanthine to the effects associated with inosine should not be underestimated). Hunter et al. (1990) [Hunter et al., 1990: (http://www.ncbi.nlm.nih.gov/pubmed/2345757)] found that elevating uric acid in the blood of rats decreased the density of A1 adenosine receptors in the striatum, a site of action of caffeine. In contrast, caffeine upregulated the A1 adenosine receptor density. This basically shows that uric acid elevations decreased the tolerance to caffeine by downregulating the chronic caffeine-induced upregulation of A1 adenosine receptor density [caffeine is a nonselective adenosine receptor antagonist, but its acute blocking effect on (antagonism of) A1 adenosine receptors figures prominently into its stimulant effects]. Thus, in a person who has never taken caffeine, the antagonism is robust, but the receptor density becomes gradually upregulated in response to the presence of the antagonism by caffeine. Increases in extracellular or intracellular uric acid may oppose that, but Hunter et al. (1990) found that uric acid was not a direct A1 adenosine receptor antagonist at physiologically-relevant concentrations. One mechanism could be feedback inhibition of xanthine oxidase activity [cited as reference 16 in Kroll et al., 1992: (http://www.ncbi.nlm.nih.gov/pubmed/1539702)]. I've never heard anyone talk about that mechanism, and it could be really important for understanding purine metabolism. There's research showing that the Ki for the feedback inhibition of xanthine oxidase by uric acid (urate) may be as low as 200 uM, a physiologically relevant concentration. This could spare ATP, given that xanthine oxidase is an ATP-consuming reaction and consumes reducing equivalents. I saw that the articles showing feedback inhibition of xanthine oxidase by endogenousl purines and purine-based drugs haven't been cited very many times, but, if that effect occurs in humans, it would be a really important mechanism, in my opinion. An excess of intracellular urate can obviously be detrimental, and one would want to discuss any of this with one's doctor and have one's uric acid checked. There's evidence that urate can inhibit glycogen phosphorylase, and caffeine can also inhibit glycogen phosphorylase. That would be counterproductive to any supposed therapeutic effects, and the levels of plasma urate at which those undesirable effects might begin to occur are not well-known. But there's research showing, for example, that hyperuricemia induced by excessive inosine supplementation (in a study in athletes whose urate levels were already high-normal) can worsen exercise performance, and inhibition of glycogen phosphorylase could account for that.