Proposed Mechanism for the Peroxynitrite-Mediated Oxidation of Uric Acid to Allantoin

God, this "Marvin" can be a real ditty to use and can still take a little time, even though I guess it is easier than the other one for this type of thing. This shows two one-electron oxidations of uric acid by ferryl heme (just because I couldn't think of another oxidant I could show the reactions of, easily) and then a series of two-electron oxidations of the products, yielding allantoin. These are proposed mechanisms that I've tried to piece together. The last reaction is shown as a two-electron oxidation, but it wouldn't really be possible in that step. Oxygen would only have 6 electrons after that. Peroxynitrite can, actually, act as either a one- or two-electron oxidant, but not in that case. I didn't feel like showing another one-electron oxidation, assuming that peroxynitrite can even bind to that carbon. The intermediates are, however, known to be intermediates in the oxidation of uric acid by peroxynitrite.

Psychedelic Mechanism for the Liberation of Nitric Oxide From a Substituted Acetohydroxamate

These articles [Samuni et al., 2009: (http://www.ncbi.nlm.nih.gov/pubmed/19447172); Gantt et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16681389); King, 2004: (http://www.ncbi.nlm.nih.gov/pubmed/15304249)] are relevant to the psychedelic mechanism shown below, in which the oxidation of a substituted acetohydroxamate (a "secondary" hydroxylamine) to its nitrosonium/oxoammonium species allows for the activation of the oxoammonium species toward a "modified Cope elimination," through a hydrogen abstraction. The third step is actually consistent with the mechanism proposed by King et al. (2004) for the formation of nitric oxide from an "unsubstituted" acylhydroxamate (a.k.a. a "primary" or terminal hydroxylamine), and King et al. (2004) argued that only those compounds with a terminal hydroxylamine moiety could serve as nitric oxide or nitrate donors. But Samuni et al. (2009) found that suberoylanilide hydroxamate, a substituted acylhydroxamate (a secondary hydroxylamine) histone deacetylase inhibitor, could serve as a nitric oxide donor, albeit by an unknown mechanism. It's interesting that a major class of histone deacetylase (HDAC) inhibitors are hydroxamate compounds that are thought to inhibit HDAC enzymes by chelating the zinc ions that are coordinated by amino acid residues on or near the active sites of the enzymes. The hydroxamate then "stays" there or something like that. But Gantt et al. (2006) found evidence and cited research to indicate that iron(II), not Zn2+, is required for the catalytic activity of HDAC enzymes. There's a lot of research showing that HDAC inhibitors are neuroprotective, and so a decrease in the availability of nonheme iron(II) to HDAC enzymes could serve an indirect, HDAC inhibitory effect, even if Zn2+ is, in fact, the metal cofactor. Or, either Zn2+ or Fe(II) could serve as the cofactor, as is the case for other enzymes, etc. The activities of HDAC enzymes can be decreased by various antioxidants, and there are "fringe" interactions through which HDAC activity can regulate HIF-1alpha expression, etc. Those are some "far out" things, in any case, that are lacking in mass appeal, probably. This is one of the last obnoxious heme drawings I'll probably be able to come up with, for the time being, as this stuff winds down. I don't know what would happen to that reactive alkene product of the "modified Cope elimination." The actual nitrogen-containing species would probably be something else, or the product I've shown would react to form something else.

Use of the Nernst Equation to Estimate the Spontaneity of a Redox Reaction (or, Here, the Reduction Potential for a "Composite" Half-Reaction)

This is not likely to be of interest to many people, but it's useful as a crude way of assessing the potential for a given reductant to reduce another species (which functions as the oxidant in the forward reaction). I was just realizing that one could also use the Nernst equation to estimate the upper or lower boundary for the reduction potential (an unknown, which is E0(sub1)) of the half-reaction for a reductant, as long as one knows that the overall reaction is occurring (deltaE > 0). In this article [Schafer and Buettner, 2001: (http://www.healthcare.uiowa.edu/corefacilities/esr/education/2003/appendix/FRBM-Redox-2001.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/11368918)], the authors discuss the use of the Nernst equation (see below) to calculate deltaE for the reduction of a given species (Ox2, which in this case is ferryl heme) by a given reductant (in this case, a hydroxamate-based drug). In one article, the authors found that the rate constant was half-maximal at a ratio of reductant to ferryl heme of about 20:1 (200 uM reductant/10 uM ferryl heme), and [Hirst and Goodwin, 2000: (http://www.jbc.org/content/275/12/8582.full.pdf+html)(http://www.ncbi.nlm.nih.gov/pubmed/10722697?dopt=Abstract)] found that, until the H2O2 was consumed, the "initial steady-state" ratio of ferryl to ferric heme, for an in vitro system in which a cytochrome c peroxidase enzyme was functioning catalytically (the enzyme is "cycling" and has a supply of H2O2 or O2), was, at a maximum, ~0.6 (Hirst and Goodwin, 2000). Koppenol and Liebman (1984) [Koppenol and Liebman, 1984: (http://pubs.acs.org/doi/abs/10.1021/j150645a024)] estimated that the redox potential for the ferryl heme/ferric heme ["compound II/metmyoglobin" (Koppenol and Liebman, 1984, p. 100)] redox couple is about +990 mV (+0.99 V). E0(sub1) is the reduction potential for a "composite" of these two reduction reactions:

RNO(rad) (nitroxide) + e(-) ---> RNO(-) (hydroxamate)
RN+=O (nitrosonium) + e(-) ---> RNO(rad) (nitroxide)

That's the convention for writing the half-reactions. It's oxidant + n(electrons donated by the reductant to the oxidant) ---> reductant (Schafer and Buettner, 2001). This would work more effectively if there were only one possible reaction, instead of two, but it's still potentially useful. Some people approximate deltaE and say that deltaE = E0(sub2) - E0(sub1). E0(sub2) = (the reduction potential or redox potential for the compound or species that's being reduced in the forward reaction), and E0(sub1) is the reduction potential for the species being oxidized in the forward reaction shown below. That approximation gives slightly different numbers, but the numbers are sort of similar and can sometimes be similar enough to get a sense of things. The Nernst equation takes into account the stoichiometry (numbers of species taking part in the half reactions and overall reactions), and that takes into account entropy (as in GSSG ---> 2GSH, forming two molecules from one), etc. The Nernst equation also takes into consideration the temperature (in the 59.1 number) and concentrations. High concentrations of one species can sometimes affect the likelihood that deltaE will be greater than 0, meaning that the rxn will be spontaneous (as in E greater than 0 for an electrochemical cell). The less than/greater than symbol (blogger won't let me put those symbols in without confusing the html code or whatever, sometimes) reverses directions because it's necessary to multiply by a negative 1, as in "crayon-based third-grade math." Actually, I have to go the "crayon route" when I do math, to avoid making errors. When one plugs those numbers into the equation and applies the crayon method, one gets this:

Two-Electron Reduction of Nitrosonium Species by Glutathione

This shows a proposed mechanism for the two-electron reduction of a nitrosonium (an oxoammonium cation), derived from an acetohydroxamic acid (shown protonated in the product, despite the unlikelihood of that), to an acetohydroxylamine (a.k.a. an acetohydroxamic acid) by glutathione, to form oxidized glutathione (GSSG). The overall mechanism is similar to the mechanism shown by Arends et al. (2006) [Arends et al., 2006: (http://cat.inist.fr/?aModele=afficheN&cpsidt=17844353)] for the two-electron reduction of an oxoammonium species by an alcohol, in the presence of a base. Boese et al. (1997) [Boese et al., 1997: (http://www.jbc.org/content/272/35/21767.full.pdf+html)(http://www.ncbi.nlm.nih.gov/pubmed/9268306?dopt=Abstract)] and Li et al. (2005) [Li et al., 2005: (http://www.jbc.org/content/280/17/16594.full.pdf+html)(http://www.ncbi.nlm.nih.gov/pubmed/15695823?dopt=Abstract)] also discussed mechanisms for reactions involving nitrosothiols, and those mechanisms are likely to be applicable in this case. There's evidence that the two-electron reduction of oxoammonium species, formed by the one-electron oxidation of nitroxide radicals, is going to be the predominant reduction pathway in vivo, even (and, in fact, particularly) during hypoxia or metabolic stress. In vitro, the one-electron reduction does occur, and the two-electron reduction also may occur.

Citrate and the "Lemonade Approach"

In this article [Kelleher et al., 1996: (http://www.ncbi.nlm.nih.gov/pubmed/8836557)], Kelleher et al. (1996) found that either citrate or fructose-1,6-bisphosphate protected cultured astrocytes and also neurons co-cultured with astrocytes against death due to hypoxia. The idea was that citrate would act as a phosphofructokinase inhibitor and inhibit the excessive glycolytic flux ("hyperglycolysis") that can occur after a traumatic brain injury or in cells under extreme hypoxic stress. That inhibition of phosphofructokinase could actually help explain the alkalinizing effect that citrate supplementation produces [McNaughton et al., 1990: (http://www.ncbi.nlm.nih.gov/pubmed/2079058)], given that glycolytic activity can lead to metabolic acidosis, especially after or during hypoxia. Those authors (McNaughton et al., 1990) found that citrate increased serum bicarbonate in a dose-dependent manner, and that's not necessarily good, past a certain point. That can be harmful in the context of phosphate depletion, etc., in the sense that an increase in serum bicarbonate can reduce oxygen unloading from hemoglobin. Increases in serum bicarbonate also fairly reliably decrease serum ionized calcium by increasing the binding of calcium to albumin (by deprotonating the sites on albumin that normally bind calcium or magnesium, etc.). Kelleher et al. (1996) noted that either fructose-1,6-BP or citrate had been found, by other researchers, to decrease the ionized calcium concentrations in the media of cultured astrocytes or neurons. There's some research showing that phosphocitrate is one "mediator" that can reduce calcium influx into mitochondria, I think, and potassium or sodium or magnesium citrate(s) have been used to prevent kidney stones. Citrate normally helps to prevent calcification in the tubular fluid, and there's a lot of research on the use of magnesium in combination with citrate (to prevent kidney stones, etc.) (http://scholar.google.com/scholar?hl=en&q=magnesium+citrate+nephrolithiasis+OR+urolithiasis&as_ylo=&as_vis=0). It's interesting that Kelleher et al. (1996) found that fluorocitrate, an aconitase inhibitor, also was protective, and the authors suggested that fluorocitrate had increased citrate availability by decreasing its conversion to isocitrate by aconitase. That could lend credence to the calcium-modulating effect or another mechanism. It doesn't necessarily rule out an effect on the TCA cycle, though, even in those experiments, and there's reason to think that citrate could have some usefulness as an energy substrate (or citric acid, which is sold also but that might be too acidic or something and evidently, but maybe not surprisingly, doesn't have the alkalinizing effect that citrate has) [Sakhaee et al., 1992: (http://www.ncbi.nlm.nih.gov/pubmed/1552616)]. There's research showing that 10-15 percent of the ATP production by cells in the proximal tubules comes from citrate oxidation [cited in Mandel, 1985: (http://www.ncbi.nlm.nih.gov/pubmed/3888090)]. I'm thinking that the use of magnesium citrate could be less likely than the use of alpha-ketoglutarate salts to cause GI-related problems, given that at least two, presumably, of citrate's ionizable carboxylate groups would be chelating the magnesium, somewhat. Apparently magnesium citrate's a salt and not a coordination compound but behaves more like a coordination compound [Lindberg et al., 1990: (http://www.ncbi.nlm.nih.gov/pubmed/2407766)]. Those authors found that Mg2+ citrate forms a soluble complex and that the bioavailability of Mg2+ from it is higher than the Mg2+ bioavailability from Mg2+ oxide. I should mention that the amount of citrate in that is large. Mg2+ citrate is about 11.3 percent magnesium. So 400 mg of elemental Mg2+, from Mg2+ citrate, would also provide 3,636 mg (3.636 grams) of citrate. So it's like a citrate supplement without the sodium or lousy calcium or potassium, etc. I haven't done any reading to see what the characteristics of the pharmacology of citrate are going to be. I'd watch for that bicarbonate effect, though, because it's not necessarily going to be rosy for a person in a disease state. But there's also the fact that, as Kelleher et al. (1996) allude to, citrate is not especially abundant. I think that the flux through citrate synthase is normally relatively low, and the reason is that oxaloacetate is in great demand. Citrate is also a major precursor of or substrate used in the formation of cytosolic acetyl-CoA and could be "lipogenic" to some extent, but one could say the same thing about glucose or glutamine or any number of "citrate precursors." It could be more true for citrate, though. There's actually research showing that citrate can help prevent ketoacidosis in people taking C5 ketones [Mochel et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/15781190)]. Mochel et al. (2005) found that citrate restored ketogenesis, basically, in people who had been taking triheptanoin, a seven-carbon precursor that's oxidized to form C5 ketones (5-carbons) and acetyl-CoA (2-carbons). It's like an "adjunctive anaplerotic agent" in that context. But those types of lipid-based energy substrates have the potential to be problematic, given that acyl-CoA thioesters tend to accumulate and inhibit all sorts of mitochondrial enzymes, etc., in my opinion.

I see that "lemonade-based" therapies (and cranberry-juice-but-not-cranberry-juice-concentrate-based therapies), as sources of citrate, have been used, instead of the citrate salts, to prevent calcium oxalate kidney stones (http://scholar.google.com/scholar?hl=en&q=nephrolithiasis+cranberry+OR+lemonade&as_ylo=&as_vis=0). Someone should change the name of citrate to something that will be taken seriously in neuroscience research. Seriously. "Well, did you take the @#$%*&ng lemonade for your stroke, like I told you, or did you sip it in a straw and get little piddly, lazy-afternoon, "lazy-lemonade" doses. Did you get that great big keg of sleepy-time lemonade like I @#$---Ah, you know, what I usually tell people is to take a half gallon or don't even take a sip."

(I was going to mention that the side effects associated with alpha-ketoglutarate salts are thought to be a consequence, at least in part, of the net charge of -2 of alpha-ketoglutarate, in the physiological pH range. Mg2+ citrate could conceivably have a net charge of -1, as opposed to the -3 charge of free citrate, much as MgATP(2-) vs. ATP(4-) have different effective net charges. Mg2+ citrate could be absorbed as a "monoanion," to some extent, and then yield the trianionic citrate in the extracellular fluid or whatever, etc.)

"Quartz (Silica) Goodies"

In this article [Flythe et al., 2009: (http://linkinghub.elsevier.com/retrieve/pii/S0272638608016132)], Flythe et al. (2009) discussed a case of a person who developed silica (silicon dioxide) kidney stones after the ingestion of silica as an excipient in supplements. The stones stopped forming after the person discontinued the source of silica. There are actually lots of reports of silica urolithiasis [Ichiyanagi et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9792982); Joekes et al., 1973: (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1588389/pdf/brmedj01539-0034.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/4350443); (http://scholar.google.com/scholar?q=related:33eEpUxra-UJ:scholar.google.com/&hl=en)]. I don't think this is likely to be a big problem for a person whose kidneys are functioning normally, but it's always possible to say things like that. "A pathological disease mechanism is likely to be more pronounced in a person in a disease state." It wouldn't be very difficult to reduce the silica intake from supplements, if one wanted to. Some supplements just use magnesium stearate as an excipient in capsules or tablets. Or one could just put the silica-containing supplements in a glass of water, let the insoluble precipitate settle (silica and calcium silicate and other silicates are not very soluble in water), and drink the water but not the silica (or pour it through a coffee machine filter--I don't know--I'm not a silica chemist, but the easier thing would just be to take some brand with no silica). From what I can tell, however, the issue is not the solubility of the silica as much as it is the fact that 1-5 percent of some forms of colloidal silica can evidently be absorbed [see last page of Jugdaohsingh et al., 2000: (http://www.ajcn.org/cgi/content/full/71/4/944)(http://www.ncbi.nlm.nih.gov/pubmed/10731501)]. That means that someone can tell a person all day that silica is insoluble, but it doesn't matter. A colloidal suspension contains, by definition, an insoluble substance that's suspended in the water or other medium (it's not called a solvent because the substance isn't being solvated). A colloid is a suspension and isn't a solution. Milk is partly a colloidal substance and is basically a lot of highly-dispersed proteins and other constituents that are not solubilized. An emulsion could be called a colloidal suspension. So if the insoluble portion can be absorbed into the bloodstream or extracellular fluid in the intestinal tract, at least, it doesn't matter, in my opinion, that it can't enter solution. It's beside the point. I looked at a few articles, and it sounds like a real mess, in the sense that one doesn't even know what "form," as in oligomeric polymers or monomers, as discussed by Jugdaohsingh et al. (2000), the silica is being supplied in.

It actually doesn't sound good to me, given that silica, in vitro, can apparently promote collagen "mineralization" (i.e. soft-tissue calcification) [see discussion by Yendt, 1973: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1946599&blobtype=pdf)]. Most of the reports of autoimmune diseases, in association with silica "exposure," have been in people who have inhaled silica dust (it causes a granulomatous disease, basically, in the lungs, by activating the macrophages that try to phagocytose the silica crystals and can't break them down and go wild, releasing pro-inflammatory cytokines, etc.). However, Parks et al. (2002) [Parks et al., 2002: (http://service004.hpc.ncsu.edu/toxicology/websites/courses/TOX705/papers/Parks%20et%20al%202002.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/12124868)] cited research (reference 16) that had shown that silica ingestion could activate macrophages in animals (meaning that the expression and release of pro-inflammatory cytokines would be increased, etc.). It obviously can be absorbed. If it weren't absorbed, one wouldn't find cases of "pure" silica-containing kidney stones in people. Even if the issue were about solubility, the GI tract is not a system at equilibrium. If 0.5 percent or whatever of a given amount of silica is absorbed (because it's soluble) in 60 minutes, say, then 0.5 percent of the remaining amount can be absorbed over the next 60 minutes or 30 minutes or whatever. Just look at the shocking case of inulin, which is still being sold [(http://hardcorephysiologyfun.blogspot.com/2009/01/disturbing-articles-on-inulin.html); (http://hardcorephysiologyfun.blogspot.com/2009/01/note-on-inulin.html)]. Here's another one showing inulin can be absorbed through the colon in rats [Ma et al., 1995: (http://www.ncbi.nlm.nih.gov/pubmed/7806033)], not just the small intestine. I'm not sure what to say, except "that's very serious business," in my opinion. It's problematic, in my opinion, to make safety determinations that are based solely, evidently, or almost solely on the supposed insolubility of a substance or on some flimsy notions about the "inviolability" of the "brush border" of the intestinal tract. I'm joking somewhat, but oh my God. It's a nonspecific immune adjuvant (Parks et al., 2002). I used to think it was essential, too, but I'm thinking that the standard "fact" about silica essentiality (that every 100th carbon, or something similar to that, in glycosaminoglycan chains is a silicon atom and that that's evidence of essentiality) isn't evidence of essentiality. That's just my opinion, but it sounds like it might just be precipitating in the extracellular matrix. Or it might be the case that some monomeric silica is being inappropriately incorporated into some of the GAG polymers? Now that I think about it, the whole thing sounds far-fetched. I doubt silica is all that harmful, but it seems like it's one of those things, like aluminum, that may not be the sole cause of a disease (such as Alzheimer's) but just shouldn't really be in foods, in my opinion. There's research showing that aluminum can exacerbate iron-mediated lipid peroxidation, for example. The FDA should have removed aluminum from the food supply 50 years ago, but that's just one of those FDA-associated horrors.

Reduction of Ferryl Heme by a Nitroxide Radical

This shows a supposed mechanism for the reduction of ferryl heme (a "compound-II-like" species) by a nitroxide radical, thereby forming the oxoammonium cation form of the nitroxide. I was going to use a single bond alongside a dative bond to show the donation of both electrons to a p(sigma)-d(sigma) bonding or antibonding orbital by oxygen, as suggested by Everse (1998) (see yesterday's postings), but the program wouldn't let me do that. I added a partial (delta) symbol to the formal charges instead, to try to convey the uncertainty about the electron configuration.