Competitive Inhibitory Effects of Vitamin B6 and Vitamin B3 on NAD+ and PLP Formation; Narrow and Variable Therapeutic Margin for Vitamin B3 (and B6)

I was remembering that vitamin B6 and vitamin B3 are very structurally similar, and researchers have found that each one is capable of inhibiting the biosynthesis of the other's coenzymes. The relevance of this is that, in my opinion, a decrease in the dosage of B6 has the potential to augment the effects of B3 and vice-versa, and this importance of the ratio of B3 to B6 is potentially significant. Niacinamide and niacin are the two most-commonly supplied forms of vitamin B3, but I'm mainly discussing the effects of niacinamide, here. Niacin has other effects on lipid metabolism that niacinamide doesn't have. I'm going to refer to niacinamide as "B3" because I'm tired of typing out the long names.

I don't feel like discussing all the potential problems with high doses of B3, but B3 can, in my opinion, produce effects that are consistent with either poly(ADP)-ribose (PAR) accumulation, resulting from the utilization of B3-derived NAD+ as a substrate for poly(ADP)-ribose polymerase and other enzymes participating in ADP-ribosylation [Hassa et al., 2006: (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1594587)(http://www.ncbi.nlm.nih.gov/pubmed/16959969)], and the associated PRPP and ATP depletion or with increases in iNOS activity, etc. It can cause thrombocytopenia [Rottembourg et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16316377)], which could be a result of the hypophosphatemia that it's also been shown to cause [Muller et al., 2007: (http://cjasn.asnjournals.org/cgi/content/full/2/6/1249)(http://www.ncbi.nlm.nih.gov/pubmed/17913971); Takahashi et al., 2004: (http://www.ncbi.nlm.nih.gov/pubmed/14871431)], and also liver dysfunction [many, many references, including probably those referring to "pruritus" from niacinamide, refer to liver dysfunction from high doses of niacinamide and niacin (cholestasis commonly causes pruritus)], either by interfering with PLP formation or depleting SAM-e in the formation of N-methylniacinamide or by increasing iNOS activity or NADPH oxidase activity, etc., etc. The thrombocytopenia appears to require rather high doses, such as 1000 mg/d (Rottembourg et al., 2005), but I wouldn't assume that any dose, above some minimal dose, is absolutely not going to cause problems. (Nicotinamide is the same thing as niacinamide.) On the other hand, B3 deficiency can cause fatty liver disease. But I generally think the effects of B3 on iNOS or PARP or NADPH oxidases can get out of control very quickly, and it's sort of like vitamin B2 and ubiquinone in that regard, in my opinion. There's much more of a rationale for using somewhat higher dosages of, for example, vitamin B5, vitamin B1, and biotin, in my opinion. But even those cofactors can lower free fatty acids excessively, as in the case of vitamin B5, or produce effects, just by their normal mechanisms, that are not always going to be desirable, in my view. But they don't really have the potential to participate in these wild, redox cycling reactions that vitamins B2 and B3 (and coenzyme Q10) can, in my opinion, participate in and facilitate.

So there's a narrow dosage range (I would define a crude, therapeutic dosage range for niacinamide as 25-75 mg/d or something, but it's possible that most of the benefits would begin to plateau at doses lower than that or at the lower end of that range), and there can be a danger in, for example, reducing the dose of B6 and finding that some aberrant or undesirable effects occur. These are all just my opinions, of course. One could erroneously conclude that the effects of a decrease in the B6 dosage are "bad" because of the B6 reduction. In reality, the "bad" effects might merely be the result of a disinhibition of the biosynthesis of NAD+ from B3, resulting from the absence of such a pronounced inhibitory effect of pyridoxine or pyridoxal or PLP on nicotinamide phosphoribosyltransferase activity, etc.

People are constantly drawing inappropriate conclusions about B3 metabolism in the literature. For example, the absence of a decrease in NAD+ levels does not necessarily mean that PAR levels have not been increased in response to exogenous B3. The B3 moiety of NAD+ can be recycled (niacinamide is the main product of the PARP reactions), but this recycling could, in my opinion, amount to a kind of ATP and PRPP depleting futile cycle. PARP contains ADP-ribose but does not sequester the actual nicotinamide (B3) moiety of NAD+, but a small increase in the pool of available, recyclable nicotinamide could conceivably waste a lot of adenine nucleotides and PRPP and ATP in the *acceleration* of ADP-ribosylation reactions. NAD+ is also a cofactor of iNOS and other NADPH oxidase enzymes, and extra B3 could just augment the formation of excessive iNOS-derived nitric oxide and produce other reactive oxygen species, in my opinion. NO (nitric oxide) also activates PARP activity, etc. People seem to think that the iNOS protein concentration and activity, in a given tissue, cannot be elevated unless a person is septic or falling on the floor from some overwhelming inflammatory disease, but this is not the case, in my view. Of course, if one thinks that NAD+ levels are going to be maximized in response to an intake of 0.5 mg per day of B3, because the National Research Council says so (I forget what it's called), then one also isn't going to be able to understand the dose-response effects of B3.

These interactions between B3 and B6 have been researched in the context of "pellagra," which is defined as a B3 deficiency disease but that can actually result from either B6 or B3 deficiencies or both. The most well-known effect is the competitive inhibition of pyridoxal kinase by niacin or niacinamide or both, and this can cause pellagra-like photosensitivity (some of the kynurenine intermediates are, apart from the porphyrins, the only known endogenously-produced photosensitizing compounds) and other effects by interfering with the B6-dependent metabolism of tryptophan. These interactions can be complex, because B6 depletion disrupts the metabolism of tryptophan to niacin (niacin can be made from tryptophan in humans, in the so-called "kynurenine pathway"). This can cause intermediates in the kynurenine pathway to accumulate, and many of these can inhibit pyridoxal kinase, the enzyme that forms PLP. That further deranges the kynurenine pathway, etc. Pyridoxal kinase is inhibited by 3-hydroxykynurenine, 3-hydroxyanthranilate, xanthurenate (i.e. xanthurenic acid), and picolinate (i.e. picolinic acid) [Takeuchi and Shibata, 1984: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1153685&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/6466295)]. That article is actually good and may help explain some of the reports of adverse effects from free-form L-tryptophan, in my opinion. I've discussed that in past postings, but Takeuchi and Shibata (1984) discuss the very high Km values for the bindings of substrates to some of those kynurenine-pathway enzymes, etc. Some of the effects of "B3 pellagra" are, obviously, just caused, proximally, by NAD+ depletion. Essentially all niacinamide is thought to be initially converted into NAD+ in vivo, but niacin is metabolized differently. I forget the precise differences, but niacin causes hypolipidemic and vasodilating ("flushing") effects by increasing prostaglandin production (by some mechanisms that I forget). But the point is that in "B3 pellagra," there isn't enough quinolinic acid available for niacin and NAD+ synthesis (see Hassa et al., 2006). As a result, more tryptophan is diverted down the kynurenine pathway that converts tryptophan to quinolinic acid, and this increases the turnover of the PLP and causes those intermediates to build up and further deplete PLP (by decreasing its formation), etc. Here are a couple other articles(not great examples) that discuss some of these interactions [Darvay et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10354170); Siniscalchi et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16039138)]. Some drugs, such as theophylline in high doses, inhibit pyridoxal kinase also, etc.

Report of Liver Dysfunction or Damage Associated With Creatine/Whey Protein Ingestion: Potential Relation to the Urea Cycle

This article [Whitt et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18452122)] discusses a case of reversible liver dysfunction/damage associated with creatine intake. I can't get the full text of the article at the moment, but there's also a study showing liver damage in animals on high-dose creatine [Tarnopolsky et al., 2003: (http://ajpregu.physiology.org/cgi/content/full/285/4/R762)(http://www.ncbi.nlm.nih.gov/pubmed/12959920?dopt=Abstract)]. I do think it might be possible for creatine to produce liver dysfunction at excessive dosages, in the long term, but that's just my opinion. The articles on the "alternate urea cycle" that occurs in people with chronic renal failure suggest that creatine or other guanidino compounds that have sometimes been shown to accumulate, at relatively low levels in humans taking 5 grams/d of creatine [Derave et al., 2004: (http://jap.physiology.org/cgi/content/full/97/3/852)(http://www.ncbi.nlm.nih.gov/pubmed/15107411?dopt=Abstract)], could inhibit urea cycle enzymes or interfere with the transport of urea cycle intermediates, etc., and produce liver dysfunction, in my opinion. This doesn't mean that creatine is "toxic" or "bad" but just means that there tends to be a therapeutic dosage range for a physiological substrate, such as creatine, and then a dosage range across which derangements in the transport or utilization of substrates with similar structures may begin to occur. That's just my opinion, but I discussed research, in a past posting, showing that high levels of glutamine, for example, could reduce endothelial nitric oxide production from eNOS, and the effect was thought to be due to the inhibition, by glutamine, of citrulline uptake or reutilization [Kawaguchi et al., 2005, cited and discussed here: (http://hardcorephysiologyfun.blogspot.com/2009/03/inhibition-of-nitric-oxide-dependent.html)]. Citrulline is a urea cycle intermediate, and so it's conceivable, in my opinion, that abnormally-high levels of glutamine or a guanidino compound derived from the transamination of arginine could, in the liver, interfere with the metabolism of citrulline in the urea cycle, etc. Glutamine, citrulline, and creatine are structurally similar, to some extent, and there's a lot of research showing competition for transport among those compounds or competitive inhibitory effects on enzymes, etc. I don't have time to get into the research on guanidinosuccinate, which has been shown to decrease in response to creatine supplementation, or argininic acid and the other guanidino compound that can accumulate during creatine supplementation (Derave et al., 2004, cited above). This could be another reason to consider using the lower end of the dosage range of creatine monohydrate used in clinical trials (~3-10 grams/d), as discussed previously, and to discuss this with one's doctor.

Research on the Use of Creatine Monohydrate as an Adjunctive to Prescription Antidepressants

These are some more articles discussing the use of creatine in combination with prescription antidepressants or medications to treat Parkinson's disease [Roitman et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17988366); Amital et al., 2006: (http://ajp.psychiatryonline.org/cgi/reprint/163/10/1840-b.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/17012702); Bender et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/17030762)]. Amital et al. (2006) researched the effects of creatine at 3 grams per day, for one week, and then 5 grams per day, for four weeks (and then also for eight weeks after the end of the trial), in a person who was being treated with prescription medications for post-traumatic stress disorder and depression with fibromyalgia. The authors found that, during the course of the trial, the person's scores on the Hamilton Depression Rating Scale had decreased, indicating that the creatine had evidently produced an antidepressant effect in the person, and that the person's symptoms of fibromyalgia had improved. Roitman et al. (2007) found that the full antidepressant effect in the patients had required four weeks to emerge, but the greatest reductions in the Hamilton Depression Rating Scale had occurred within the first two weeks in most of the people. Roitman et al. (2007) also found that some people experienced a greater improvement in mood from 3 grams/day of creatine than from 5 grams/day.

Bender et al. (2006) used 4 grams a day of creatine for two years (after an initial phase of 20 grams per day for 6 days and then 2 grams per day for six months), in a randomized, placebo-controlled trial and found that the people who had taken creatine had not had to increase the dosages of their dopaminergic medications for Parkinson's disease to the extent that the people taking the placebo had had to increase their dosages, and that effect was statistically-significant. The other main result of the trial was the finding that creatine had produced a statistically-significant reduction in symptoms of depression, as measured by the "score" on Item 3 of Part I of the Unified Parkinson's Disease Rating Scale (UPDRS). Incidentally, I don't think the "loading dose" concept of creatine makes much sense, especially given the potential for high-dose creatine to produce plasma and extracellular fluid volume expansion in some people [Powers et al., 2003: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=155510&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/12937471)]. To the extent that that effect would occur more with high-dose than with low-dose creatine, one would expect the body to be less able to adapt or compensate to that effect in the context of a "loading dose." But that's just my opinion. Obviously, one would want to discuss this with one's doctor before using creatine or any supplement, particularly given that Roitman et al. (2007) found that, in one of the ten patients, creatine produced transient improvement in the person's mood and then worsened it. Roitman (2007) also found that creatine produced mania or hypomania in the people in the trial who had been diagnosed with bipolar disorder.

In my opinion, creatine would be more effective in this context in combination with adenosine monophosphate/triphosphate or guanosine monophosphate or both, for complex reasons. I think it's a really important point, but it's just my opinion. I could explain my reasoning in more detail, but I'd have to cite a lot more articles and can't do that right now. I discussed some of the rationale for the combination in a past posting (http://hardcorephysiologyfun.blogspot.com/2009/03/creatine-cr-phosphocreatine-pcr-and.html), and I discussed some of the research and details on orally-administered guanosine monophosphate and adenosine monophosphate or adenosine triphosphate in this posting (http://hardcorephysiologyfun.blogspot.com/2009/03/adenosine-and-guanosine-in-animal.html). Other approaches that might potentiate the effects of creatine, in my opinion, would be the use of methylcobalamin at ~5 mg/day or something (with the intent of disinhibiting creatine kinase enzymes and tricarboxylic acid cycle enzymes by reducing methylmalonic acid levels), the use of L-methylfolate or levoleucovorin in combination with methylcobalamin (with the intent of reducing the accumulation of sarcosine, which may be produced from high-dose creatine, given that folate depletion can compromise the metabolism of sarcosine and dimethylglycine and betaine, as I've discussed in the context of research cited in past postings), and the use of adenosine and guanosine to elevate serum uric acid to high-normal levels, under a doctor's supervision (with various "purposes" in mind, including the disinhibition of creatine kinase, which is inhibited by peroxynitrite, through the scavenging of peroxynitrite by uric acid) [discussed and cited here: (http://hardcorephysiologyfun.blogspot.com/2009/03/creatine-cr-phosphocreatine-pcr-and.html)]. Poortmans et al. (2005) [Poortmans et al., 2005: (http://www.ncbi.nlm.nih.gov/pubmed/16260971)] discussed the fact that the elevation of urinary methylamine and formaldehyde, in response to high-dose creatine administration, could have resulted from the metabolism of some of the creatine to sarcosine, evidently by the creatinase enzymatic activity in microorganisms in the G.I. tract, if memory serves (humans don't express a creatinase enzyme, as far as is known). Sarcosine can be metabolized to methylamine by sarcosine reductase or to formaldehyde and glycine, by sarcosine oxidase, and methylamine can be metabolized to formaldehyde and ammonia by semicarbazide-sensitive amine oxidase (SSAO) (Poortmans et al., 2005). Formaldehyde can then be converted into formate, and the activities of the folate cycle enzymes, which are dependent upon adequate pools of reduced folates, such as L-methylfolate or levoleucovorin, and vitamin B12 (i.e. methylcobalamin), are important for the normal metabolism of formate.

Modulation of Nitrergic Transmission by Methylcobalamin and Hydroxocobalamin or Increases in Observed Km's via Sequestration of Endogenous Cobalamins?

This article is really important in the context of the supposed therapeutic uses of high-dose (I'm talking about very high doses, such as the bizarrely-high doses used in research in the treatment of amyotrophic lateral sclerosis (ALS), not the high doses that are used to overcome endogenous inhibitors of B12-dependent enzymes in people who do not have neurodegenerative diseases, etc.) methylcobalamin (MeCbl) or hydroxocobalamin (OHCbl) (forms of vitamin B12 that are not "equivalent." in terms of their effects, to cyanocobalamin) [Oh et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10071959)]. The authors found that extracellular OHCbl scavenged (bound, either reversibly or irreversibly) nitric oxide (NO) and thereby decreased the release of glutamate in response to the activation of NMDA receptors by exogenous N-methyl-D-aspartate (a drug ligand for NMDA glutamate receptors). As far as I can tell, this is the only paper that has provided reasonably-direct evidence of the modulation of glutamatergic transmission, by NO scavenging or "NO buffering," by OHCbl. Other researchers have shown that high concentrations of methylcobalamin (usually 10 uM) can produce either excitation or attenuation of glutamatergic transmission [(http://scholar.google.com/scholar?num=100&hl=en&lr=&q=methylcobalamin+NMDA+OR+glutamate); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=hydroxocobalamin+NMDA+OR+glutamate); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22nitric+oxide%22+hydroxocobalamin+OR+methylcobalamin)], and the modulation by MeCbl or OHCbl of nitrergic neurotransmission has been shown in the contexts of other neurotransmitter systems, etc. [Colpaert and Lefebvre, 2000: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1571952&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/10725269)]. Colpaert and Lefebvre (2000) discuss the capacity of OHCbl to either reversibly or irreversibly bind NO to form nitrosocobalamin (NOCbl) and other cobalamins (superoxocobalamin, formed by a reaction of superoxide with Cbl, can also bind NO, and the character of the binding essentially depends on the oxidation state of the cobalt atom of Cbl. These might be some other articles by that group (http://scholar.google.com/scholar?q=Colpaert+cobalamin+OR+hydroxocobalamin+OR+methylcobalamin&num=100&hl=en&lr=).

The paper by Oh et al. (1999) is a really important paper, and it's conceivable that lower concentrations of OHCbl or MeCbl would produce some of this effect. This NO-buffering effect of either MeCbl or OHCbl has been shown in many other articles, and the most commonly-observed net effect of MeCbl or OHCbl is essentially to prolong the action of NO while reducing the amplitude of the initial response, such as the excitatory postsynaptic potential or calcium influx or smooth muscle cell contraction or relaxation. Colpaert and Lefebvre (2000) discuss that type of thing.

A lot of authors have written articles about the NO-scavenging effect of methylcobalamin and the potential relevance to inflammatory conditions that have been associated with "B12-responsiveness." I tend to think these effects on glutamatergic transmission, resulting from NO-buffering (as opposed to, for example, a B12-induced disinhibition of the tricarboxylic acid cycle (TCA cycle) in response to a decrease in intracellular or intramitochondrial methylmalonic acid (MMA) levels), would mainly occur at very high doses (much higher than those used in all but a few clinical trials). I say that because the concentration used in those articles has typically been 10 uM, which is much higher than the serum or extracellular fluid concentrations of Cbl's that are seen normally, even in response to 250-500 ug of MeCbl per day, given parenterally. I showed the serum B12 values for very high dose MeCbl in a past posting, and the levels were only about ~34.2-36.7 nM (http://hardcorephysiologyfun.blogspot.com/2009/01/unanswered-questions-about.html). But I do think that endogenously-produced NOCbl or glutathionylcobalamin could increase the observed/effective Km for the binding of 5'-deoxyadenosylcobalamin to methylmalonyl-CoA mutase (MMM) as a cofactor (or the observed Km for the binding of MeCbl to methionine synthase).

It's remarkable that I've never seen a measurement or estimate of the extent to which endogenously-produced NOCbl or other species could increase the effective Km's for the binding of Cbl-derived cofactors (MeCbl and AdoCbl) to their respective enzymes. Some of the data from cell culture studies hint at that effect [similar to the so-called "arginine paradox," in which increases in extracellular arginine can increase the activities of nitric oxide synthase (NOS) enzymes at concentrations up to 500 uM, which is much higher than the Km, for arginine binding to endothelial NOS, derived from research (research that failed to take into account the pronounced in vivo inhibition of NOS enzymes by ADMA and N(omega)-monomethylarginine and other endogenous NOS inhibitors, etc.]. It would be hard to measure the binding of endogenously-produced NO to Cbl's, though, because the binding can be reversible. I think it could be done, though. Peters et al. (1983) [Peters et al., 1983: (http://jn.nutrition.org/cgi/reprint/113/6/1221.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/6854414)] found that propionate uptake (and, by extension, the propionate oxidation rate) increased as the liver cobalamin contents of sheep increased up to 250 ng/g ww. That works out to about 300 nM (250 ng/g ww x 1000)/(1355.38 x 0.615) (http://hardcorephysiologyfun.blogspot.com/2008/12/cell-biology-conversion-factors-for-ngg.html) as an intracellular concentration of total cobalamins in the livers of the sheep. Given that AdoCbl constitutes ~72.8 percent of the total cobalamins in the livers of humans [Yamada et al., 2000: (http://jn.nutrition.org/cgi/reprint/130/8/1894)(http://www.ncbi.nlm.nih.gov/pubmed/10917899?dopt=Abstract)] (and assuming these percentages and Km values are comparable for sheep and humans, an assumption that might be incorrect), the maximal rate of propionate uptake (and, by extension, oxidation) may not occur until the intracellular AdoCbl concentration is ~218 nM. That's about 3.5-4 times the Km for AdoCbl binding to MMM in the human liver. The Km values for AdoCbl binding to human wild-type MMM have been found to be 50 nM or 62.5 nM in the human liver or in human fibroblasts (http://hardcorephysiologyfun.blogspot.com/2009/01/km-values-for-adocbl-binding-to-mmm-and.html), and the provision of B12 from standard diets in animals or humans, in the absence of supplementation, is likely to produce intracellular concentrations of AdoCbl that are far below the Km for AdoCbl binding to MMM (http://hardcorephysiologyfun.blogspot.com/2009/01/methylcobalamin-and-other-forms-of.html). It's possible that the sheep Km is higher, but I still tend to think endogenous inactivation or "sequestration" of Cbl's (as glutathionylcobalamin or NOCbl) could explain the ongoing findings that higher doses of MeCbl sometimes lower homocysteine levels more effectively than more commonly-used dosages. Also, ischemia can upregulate MMM expression and could increase the effective/observed Km values. When one considers that NO itself (and undoubtedly other endogenous inhibitors) can inhibit MMM activity, it's not unreasonable to think that the observed Km values for Cbl-dependent enzymes might be higher than the strictly-defined values found in the absence of inflammation during in vitro experiments, etc.

Arginine, Uric Acid, and Peroxynitrite in Neurodegenerative and Psychiatric Conditions

This article [Xia et al., 1996: (http://www.pnas.org/cgi/reprint/93/13/6770.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/8692893)] is one of many showing that depletion of intracellular arginine tends to increase peroxynitrite formation by multiple mechanisms. When nitric oxide synthase (NOS) enzymes are not occupied by arginine or are competitively inhibited by asymmetric N(G),N(G)-dimethylarginine (ADMA) or by other methylarginines, which are inhibitors of NOS enzymes that are produced normally during the breakdown of proteins, the NOS enzymes produce superoxide and can also produce NO and superoxide at the same time. The NOS-derived NO and superoxide tend to react to form peroxynitrite. The depletion of cytosolic arginine by roughly half produced a fivefold increase in the sensitivities of the cells to a cytotoxic stimulus that increased nNOS activity. Similarly, Xia and Zweier (1997) [Xia and Zweier, 1997: (http://www.pnas.org/cgi/content/full/94/13/6954)] found that arginine depletion from activated macrophages produced large increases in peroxynitrite levels, and these increases were almost entirely blocked by either 1 mM extracellular arginine or 1 mM extracellular urate (the form that uric acid is in at physiological pH values).

This is relevant to the effects of arginine and purines in the brain. I think that maintaining an adequate urate level in the cerebrospinal fluid and also intracellularly, in neurons and astrocytes, is likely to be really important for maintaining cellular energy metabolism and also for maintaining the normal nitrergic regulation of noradrenergic and dopaminergic transmission, such as through the effects of nitric oxide on NMDA receptor activation. Roitman et al. (2007) [cited here: (http://hardcorephysiologyfun.blogspot.com/2009/03/arginine-agmatine-and-nitric-oxide-in.html)] noted some of the evidence that impairments in cellular energy metabolism can be found in people with depression and other psychiatric symptoms. Phosphocreatine (PCr) levels have been shown, in research using magnetic resonance spectroscopy techniques, to be drastically depleted in the brains of people with depression, for example (cited in Roitman et al., 2007). Mitochondrial dysfunction and ATP depletion would reasonably be expected to produce PCr depletion, even in the absence of a deficit in the formation of new creatine from arginine, etc. There's actually a large amount of research showing protection by uric acid/urate against mitochondrial damage due to peroxynitrite (inactivation of complex I and mitochondrial dysfunction) (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=mitochondrial+peroxynitrite+uric+OR+urate).

The reason I didn't see those articles in the past is that I think the authors of many articles showing protective effects of urate tend to not mention multiple sclerosis or Parkinson's disease, two neurodegenerative diseases in which inosine, a precursor of urate, is being tested (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=Parkinson%27s+uric+OR+urate); (http://scholar.google.com/scholar?num=100&hl=en&lr=&q=%22multiple+sclerosis%22+uric+OR+urate)]. They would be much better off (and would, in my opinion, get substantially more robust therapeutic effects) using guanosine and adenosine monophosphates or triphosphates as precursors of urate instead of inosine, in my opinion, but that's beside the point. The research tends to be very focused in on one little area, and I've never seen those articles on mitochondrial protection, by urate, cited in the context of Parkinson's disease or MS. But the potential for the protection, by urate (or arginine), against the compromising of cellular energy metabolism, by peroxynitrite, would be very important, in my opinion, in the contexts of those and other disorders. West et al. (2002) [cited here: (http://hardcorephysiologyfun.blogspot.com/2009/03/arginine-agmatine-and-nitric-oxide-in.html)] cited research showing that peroxynitrite tends to decrease dopamine release (as in tonic, excitatory, nitrergically-mediated dopamine release), and that's obviously relevant to cognition and psychiatric conditions. I think the peroxynitrite-reducing potential of arginine would be more likely to be effective in combination with normalization of CSF and intracellular urate levels in the brain. If a person's plasma urate (blood uric acid) is already high, the person wouldn't need to do this. But the notion that any old urate level is as "good" as any other, within the normal range, is not defensible, given the overwhelming evidence, in my opinion, showing major effects across small increments in extracellular and, by extension, intracellular urate concentrations. I don't have time to go into the articles showing high intracellular urate levels, but the main idea is, in my opinion, that the use of urate as a peroxynitrite scavenger makes the use of most other antioxidants look like child's play. The concentrations of urate that scavenge peroxynitrite meaningfully, in vitro, are comparable to achievable and normal in vivo concentrations (this is not the case at all for many antioxidants). The concentrations of urate, both intracellularly and extracellularly, are much, much higher than the concentrations one is going to achieve with most antioxidants, in my opinion. Additionally, urate is less like an antioxidant scavenger of peroxynitrite than it is like a peroxynitrite "sink" and is, for the most part, excreted. It is not regnerated (doesn't need to be regenerated) by oxidoreductase enzymes but can actually be degraded, in a series of intramolecular degradative reactions (to allantoin or other molecules), upon its nitrosylation/nitration/etc. That's a really unique property that sets it apart from most other so-called "antioxidants" that consume reducing equivalents in their regeneration. I do think there's a lot of validity to the peroxynitrite-reducing effects of arginine in the context of brain disorders [Wiesinger, 2001: (http://www.ncbi.nlm.nih.gov/pubmed/11275358)], but I think that approach would work better in combination with normalization of urate levels. Obviously, one should discuss this type of thing with one's doctor.

Arginine, Agmatine, and Nitric Oxide in Psychiatric Conditions and Neuroprotection: Abbreviated Posting

This article shows that exogenous L-arginine exerted a biphasic effect on adult mice in the forced swim test, an animal model of depression [Ergun and Ergun, 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17125765)], such that low but not high doses of arginine produced antidepressant effects. The higher doses increased immobility (suggestive of a depressogenic or depressant effect), and the authors cite two other articles showing similar biphasic effects of arginine [Da Silva et al., 2000: (http://www.ncbi.nlm.nih.gov/pubmed/11117475); and Inan et al., 2004: (http://www.ncbi.nlm.nih.gov/pubmed/15006455)]. The authors also found evidence that this biphasic response can be explained in terms of "depressant" effects of high levels of nitric oxide (NO) and antidepressant effects of smaller increases in NO formation. Arginine supplementation tends to increase the production of NO, a short-lived signalling molecule that plays a major role in maintaining blood vessel dilation and is released from nitrergic neurons that provide synaptic inputs to neurons throughout the brain, including glutamatergic and dopaminergic neurons in the striatum and noradrenergic neurons in the locus ceruleus [French et al., 2005: (http://www.med.upenn.edu/taylor/pubs/french%20neurosci%202005.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/16084659)]. Most of the nitrergic neurons in the striatum are nonspiny interneurons. Ergun and Ergun (2007) cited research [Ergun et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16940926/); Harkin et al., 1999: (http://www.ncbi.nlm.nih.gov/pubmed/10395013)] that the administration of different dosages of NOS inhibitors (which, under these conditions, are thought to be exerting their effects through the inhibition of neuronal NOS (nNOS) activity and not through the inhibition of inducible NOS (iNOS), given the absence of inflammation and short-term time frame, etc.) [West et al., 2002: (http://www.med.wayne.edu/neuroscience/labs/bird/pdfs/Tony-NO-rev.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/11984858)] also produces biphasic antidepressant effects in animal models.

Some of these effects of arginine and NOS inhibitors in animal models of depression are likely to be occurring in parts of the brain other than the striatum, such as in the locus ceruleus, etc. This is a really complicated area, and the effects of arginine in animal models are mainly acute or short-term effects and would not be expected to be the same in the context of chronic stress or depression or in other situations. And the same compound or physiological process that is beneficial to one person may be detrimental to another, and that's the reason it's always necessary for people to discuss these things with one's doctor. But I think there's enough research on arginine and agmatine and nitrergic transmission to get a general picture of the dose-response relationships and some of the predominant pharmacological effects that would be expected to occur in humans (under various conditions).

The general idea is that, under conditions that are not producing acute or chronic activation of the stress response pathways in the brain (i.e. increases in the firing rates of noradrenergic neurons in the locus ceruleus, etc.), exogenous arginine at "low doses" will, in my opinion, exert its short term effects by producing NO-dependent inhibition of the glutamatergically-mediated release of noradrenaline or dopamine or both and simultaneously decrease the firing rates of noradrenergic neurons. More specifically, I think the NO-mediated decrease in calcium influx in response to NMDA receptor activation will be the type of effect that will, at lower doses of arginine, predominate over the excitatory effects of NO. Some of the effects of the acute administration of "low" dosages of arginine may also be mediated by agmatine, which is produced in the brain in large amounts in response to arginine supplementation in animals and probably also would be, in my opinion, in humans. At higher dosages, I think arginine would tend to produce enhancements in glutamatergic transmission, mediated by both increases in nNOS-derived NO and increases in agmatine levels (agmatine may release glutamate and thereby produce excitatory effects at higher dosages [Halaris and Pleitz, 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17927294)]), that could well be counterproductive to the treatment of depression or chronic stress. Researchers have found that the dosages of intraperitoneal (i.p.) L-arginine, in mice, that produce "antidepressant-like" effects in animal models of depression scale to acute, single human dosages, for a 70-kg human (http://hardcorephysiologyfun.blogspot.com/2008/12/equations-for-animal-food-intake-and.html), of 3022-6045 mg (Da Silva et al., 2000), 6045-12090 mg (Inan et al., 2004), or 1209 mg (Ergun and Ergun, 2007). Inan et al. (2004) found that low dosages of i.p. arginine (dosages that scale to i.p. dosages of 302 or 1209 mg for a 70-kg human) produced "depressant" effects and blocked the antidepressant effects of potassium channel blockers or nNOS inhibitors, but Ergun and Ergun (2007) found that only low but not high doses of arginine were consistent with "antidepressant-like" effects. It's possible to make sense of these discrepancies, to some extent, but one can't look at data from those models in excessively-rigid terms.

The NMDA antagonistic effects of arginine-derived nitric oxide (and, more indirectly, of arginine-derived agmatine) would, in my opinion, have relevance to the augmentation of conventional antidepressants or to restoring the effectiveness of some psychopharmacological strategies, but there are a lot of details that are really complicated to get into. It's fairly clear, from the literature, that nitric oxide normally produces "tonic" (meaning under baseline conditions, in conditions other than animal models of chronic stress or depression, in which the firing rates of locus ceruleus neurons are going to be increased) excitatory influence on both noradrenaline release, by noradrenergic neurons in the locus ceruleus, and on dopamine release in the striatum. For example, arginine can increase dopamine release in the striatum by enhancing nNOS-derived NO [Liang and Kaufman, 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9685635)] and can also increase the firing rates of noradrenergic neurons in the locus ceruleus, evidently by nNOS-independent glutamatergic effects (this might be due to agmatine) [Torrecilla et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17473915)]. Liang and Kaufman cite four other articles [Hirsch et al., 1993: (http://www.ncbi.nlm.nih.gov/pubmed/15335838); Lonart et al., 1992: (http://www.ncbi.nlm.nih.gov/pubmed/1425999); Strasser et al., 1994: (http://www.ncbi.nlm.nih.gov/pubmed/7533554) Zhu and Luo, 1992: (http://www.ncbi.nlm.nih.gov/pubmed/1494918)] showing the same nitrergically-mediated dopamine release in response to L-arginine, and I'm sure there are other articles showing that. That's probably partly a glutamatergic effect that's mediated by the nitric oxide-induced activation of glutamate release or inhibition of chloride influx in response to GABA-A-receptor activation, etc. But under conditions of chronic increases in the firing rates of noradrenergic neurons or pathological increases in glutamatergic transmission, more agmatine is formed from arginine and may help to limit the firing rates of noradrenergic neurons and thereby produce an "anti-stress" effect (this stress-induced increase in agmatine formation has been shown in animal experiments, in which acute stress roughly triples the tissue agmatine contents in multiple parts of the brain). In the striatum, NMDA receptor antagonism, such as by nitric oxide, can actually sensitize striatal neurons to D1 dopamine receptor activation via dopaminergic inputs from the ventral tegmental area. This can produce beneficial effects on working memory, to a point, but can then, at higher degrees of NMDA receptor antagonism, impair working memory by causing a breakdown of the organized, burst firing patterns of glutamatergic pyramidal neurons in the prefrontal cortex (in association with working memory impairment). So basal dopaminergic activity and noradrenergic activities appear to be dependent on an adequate level of NO-mediated glutamatergic activity, but the stress-induced activation of noradrenergic neurons, among other effects, could, in my opinion, narrow or even "abolish" the supposed therapeutic dosage range for something like arginine, in this context.

Incidentally, the acute oral dosages that would produce comparable effects in the brain (comparable to the i.p. dosages) would be expected to be higher than the i.p. dosages, and the increases in plasma arginine [Bode-Boger et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9833603)(http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1873701&blobtype=pdf)], following daily supplementation, can take 4-8 weeks to reach a steady state in humans [Campbell et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16928472)]. Bode-Boger et al. (1998) found that the absolute bioavailability of oral arginine in humans, in relation to i.v. arginine, was ~68 percent, and the ratio of the AUC(i.v.)/AUC(oral) was ~1.542 for dosages of 6 grams of arginine (by i.v. and oral routes). I think the ratio of the Cmax(i.v.)/Cmax(oral) (= 2.652 for arginine at dosages of 6 grams, given by i.v. and oral routes) is likely to be a more important determinant of the effects of arginine on the brain than the ratio of the AUC values, because the concentration of agmatine in the CSF (and, by extension, in the CNS intraparenchymal interstitial fluid) increases and decreases rapidly. This suggests to me that the rate of entry of arginine into the brain may importantly determine the metabolic fate of arginine. More specifically, the arginine-induced production of agmatine in the brain would be expected to be higher in response to arginine taken in the fasted state, meaning before breakfast in the morning, than in response to arginine taken between meals. The amounts of agmatine formed in the brain from exogenous arginine appear to be quite significant in primates and mice [Piletz et al., 2003: (http://www.ncbi.nlm.nih.gov/pubmed/15028571)], and I'll include my analysis of that type of data, together with my estimate of the short-term extracellular and intracellular concentrations of agmatine in response to scaled doses of arginine, in another posting. Suffice it to say that a single dose of intraperitoneally-administered arginine in a monkey (this scales to a human dose of 6176 mg arginine, given intraperitoneally) produced a peak level of 2200 nM (2.2 uM) agmatine in the CSF (up from baseline values of 46.9-181 nM). The Kd values for the binding of agmatine to I1 and I2 imidazoline receptors (IRs), producing competitive inhibition, are 700 nM (0.7 uM) and 1000 nM (1 uM), and the Kd for the binding of agmatine to alpha2-adrenoreceptors (alpha2-ARs), as an agonist, is ~4000 nM (4 uM). Clonidine produces activation of both I1-IRs and alpha2-ARs, and agmatine, to a meaningful extent, essentially produces clonidine-like effects. But the point is that the more robust activation of I1-IRs by agmatine, at extracellular agmatine levels less than the range of the 4 uM Kd value for strong alpha2-AR activation by agmatine, could reasonably be expected to augment the effect of low-level alpha2-AR activation by the arginine-induced increases in agmatine. I haven't seen much compelling evidence that meaningful antagonism of NMDA receptors occurs at extracellular concentrations of agmatine that occur under normal or therapeutic circumstances, in response to either arginine or agmatine administration. The Ki value is really high for NMDA receptor antagonism by agmatine, and the authors of one article, showing evidence of NMDA receptor antagonism in response to intrathecal agmatine (or i.c.v.--I can't remember right now and don't want to look it up), found that the effect only lasted between 10 and 30 minutes. The extracellular concentration was very high. One key point, though, is that i.v. arginine increased the CSF agmatine to a peak level that was 22 times as high as the increase in plasma agmatine. Thus, these articles that measure plasma agmatine in response to arginine are not going to be detecting the agmatine formed en masse in the CSF. I think arginine-induced agmatine participates in the arginine-induced growth hormone release, also. Additionally, I think the agmatine levels would accumulate over time intracellularly. It's rapidly transported into neurons by a polyamine transporter that transports spermine and putrescine (and presumably spermidine), and the results of the article by Piletz et al. (2003) (cited above) suggest that most of the tissue agmatine levels, at three hours post-i.p. injection, will be intracellular and not extracellular. I can't extrapolate the intracellular concentration that accompanied the large peak in CSF agmatine that occurred in the primates, but, under steady-state conditions, it's pretty clear that about 95.6 percent of a measurement of the tissue agmatine levels, in ng/g wet weight, is intracellular (with the rest being extracellular). I'll put the simple calculations up on another posting. (You can estimate this if you know, roughly, both the steady-state CSF concentration and the tissue concentration, in ng/g ww.) Finally, in the longer term, there's evidence to suggest that the inhibitory effects of arginine-derived agmatine on nNOS activity may become more significant and produce meaningful effects, by glutamatergic or other mechanisms, on noradrenergic or dopaminergic transmission. The Ki value for irreversible (noncompetitive) inhibition of nNOS by agmatine (as agmatine aldehyde) is 29 uM (Piletz et al., 2003, cited above) (the Ki value for competitive inhibition by agmatine is much higher), and the supposed accumulation of stored, intracellular agmatine could conceivably produce some gradual inhibition of nNOS activity over time. That could produce NMDA receptor antagonism, by reducing nNOS-derived NO, but the direct antagonism of NMDA receptors by agmatine seems to be an effect that, in my opinion, is unlikely to become meaningful.

In any case, this is too complicated a topic to discuss all at once. Arginine has also been shown to augment creatine formation in the brain in humans, in two articles, and in animals, and low-dose creatine (3-5 grams/d) was shown to be beneficial, in a small study, in augmenting conventional antidepressant medications in people with treatment-resistant depression [Roitman et al., 2007: (http://www.ncbi.nlm.nih.gov/pubmed/17988366)]. I think arginine would be safer for that effect [discussed here: (http://hardcorephysiologyfun.blogspot.com/2009/02/glutamate-glutamine-cycle-de-novo.html)], but one approach could be to use extremely small doses of creatine, such as 1-2 grams/d. Supposedly, diets high in red meat provide 1-2 grams of creatine/d, but I think some of those estimates of dietary creatine overestimate the amounts that people would be getting. It's interesting that Roitman et al. (2007) found that a couple of the people lost the mood-elevating effect from creatine as they increased the dosage from 3 to 5 grams per day. When they went back to the 3 g/d dosage, they benefited from it again. I think that's plausible, that there would be a dose-response relationship across very small dosage increments. The main problem, in my opinion, is not with the short-term effects of creatine but with the fact that the long-term effects can be very complicated and can become counterproductive, even in the extreme, in my opinion. The therapeutic window for creatine dosing, for psychiatric or cognitive effects [Roitman et al. (2007) cite research showing cognitive enhancement from something like 5 grams/d of creatine in normal people, and there's a vast amount of research on its effects on the brain], is probably very narrow, and the therapeutic dosages could be, in my opinion, as low as 1-3 grams/d. Also, there's the issue of the conversion of ornithine or agmatine, both derived from arginine, into putrescine and the other polyamines, and putrescine has been shown to produce antidepressant effects in animal models. The antidepressant effects that S-adenosylmethionine (SAM-e) has been shown to exert in animal models may partly be due to the SAM-e-induced elevations in the levels of putrescine and other polyamines in the brain, but, in my opinion, the effects of SAM-e have more to do with increases in the adenosine nucleotide pools in neurons and astrocytes and could be mimicked by exogenous adenosine (and guanosine). Decarboxylated SAM-e is a cofactor for spermine synthase, which converts spermidine into spermine, and spermidine synthase, an enzyme that converts putrescine into spermidine. Putrescine can be formed by the catabolism of agmatine by agmatinase or by the pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine decarboxylase. The authors of one article I discussed [Geng et al., 1995: (http://hardcorephysiologyfun.blogspot.com/2009/01/mechanisms-of-neuroprotection-by.html)] found evidence that the neuroprotective effects of vitamin B6 (pyridoxine), in cultured cells, were partially mediated by the PLP-dependent increase in ornithine decarboxylase activity (via the formation of putrescine and the other polyamines, which then can either antagonize or produce positive allosteric activation of NMDA receptors), given that ifenprodil partially blocked the PLP-mediated neuroprotection. The same modulation of NMDA receptor activation could account for the effects of changes in polyamine levels on cognitive functioning or psychiatric conditions, but research on polyamines seems to be rather chaotic and confusing for just about everyone who writes about it.

Arginine could be expected to increase both agmatine and ornithine and to increase putrescine formation from either agmatine or ornithine, whereas ornithine may not increase agmatine nearly as much and would tend to inhibit creatine formation by arginine:glycine amidinotransferase (AGAT) (the first enzyme in creatine biosynthesis that is expressed throughout the brain). There's also the issue of peroxynitrite formation by the reaction of excessive NO with superoxide or by the uncoupling of nNOS by asymmetric N(guanidino),N(guanidino)-dimethylarginine (ADMA) or N(omega)-monomethylarginine (endogenously-produced inhibitors of NOS enzymes), etc., and maintaining a high-normal CSF uric acid level, with low-dose purines, could, in my opinion, help to limit peroxynitrite formation and downregulate iNOS expression. I can't link to 100 articles in one posting, though.

Inhibition of Nitric-Oxide-Dependent Vasodilation by High Levels of Glutamine in Endothelial Cells: Relevance of Arginine Availability and Exercise

This article [Kawaguchi et al., 2005: (http://ajpregu.physiology.org/cgi/reprint/288/6/R1612)(http://www.ncbi.nlm.nih.gov/pubmed/15705802?dopt=Abstract)] shows that exogenous glutamine, without concomitant hyperammonemia (abnormal elevation of ammonia) can inhibit the dilation of cerebral arterioles (the smaller-diameter branches of the pial arteries that run along the outer surface of the brain) in rats, and exogenous arginine can block the effect, to some extent. There are lots of articles that show the same thing, and, in my opinion, this shows that elevating the plasma glutamine levels would only be expected to be beneficial up to a point. There's quite a bit of evidence, discussed by Kawaguchi et al. (2005), that the effect of glutamine is the result of its competition with citrulline for transport into endothelial cells or perivascular nerve fibers (fibers that induce nitric-oxide-dependent vasodilation). Citrulline is converted into arginine, and arginine is then used as a substrate for nitric oxide biosynthesis by eNOS. The citrulline formed as a product is then recycled, and an excess of glutamine is thought to inhibit the recycling (or the transport of citrulline into cells from the blood) of citrulline. The authors also noted that glucosamine, formed from glutamine, could have produced the impairment in vasodilation. I think only a small percentage, about 3 percent, of glutamine is converted into glucosamine in the liver, but I don't know what percentage is converted into glucosamine in endothelial cells or neurons (i.e. perivascular neurons, etc.).

Although a person could, in my opinion, conceivably help to minimize this type of effect by taking glutamine with meals (thereby preventing spikes in plasma glutamine and deliberately limiting its bioavailability) and spreading the dosages out across the day, it's important to consider the drastic reductions in plasma glutamine that can occur, for example, during and after intense exercise or in catabolic disease states. Keast et al. (1995) [Keast et al., 1995: (http://www.ncbi.nlm.nih.gov/pubmed/7845291)] found that high-intensity exercise depleted plasma glutamine levels by more than half, and more than 6 days were required for the restoration of pre-"overtraining" plasma glutamine to occur in some of the athletes. For some reason, there's been an obsessive focus on controversy about the role of glutamine depletion in the immunosuppressive effects that high-intensity exercise can have. I don't know why there has been so much focus on that aspect of glutamine metabolism, but it doesn't matter if glutamine depletion is not a major role in exercise-induced immunosuppression. By definition, someone who can exercise at high intensities is going to be fairly "healthy," and this makes the entire debate, surrounding that specific issue, seem bizarre to me. For example, Walsh et al. (1998) [Walsh et al., 1998: (http://www.ncbi.nlm.nih.gov/pubmed/9562294)] found not-very-drastic reductions in plasma glutamine following exercise (of poorly-defined intensity) but took the measurements in "well-trained" athletes. In my mind, the capacity of an athlete to replenish glutamine levels in his or her blood or muscles or heart or endothelial cells is going to be fairly robust. A major point of the hundreds of articles showing immunostimulatory effects of glutamine in trauma patients, etc., is that one cannot, in my opinion, look at the results of studies in elite athletes and then generalize or apply those results to a discussion or analysis of glutamine or cell-energy metabolism in the context of disease states.

That's unrelated to the original topic, and it's still important, in my opinion, to take seriously the possibility of a nitric-oxide-suppressive effect of excessively-high glutamine intakes, particularly in the absence of arginine. It's worth noting that significant portion of glutamine is converted into citrulline and alanine in the intestinal tract, and the blood-borne citrulline can then be converted into arginine in the kidneys or, locally, in endothelial cells. So that would tend to oppose or counteract the supposed vasoconstrictive effects of abnormal elevations in plasma glutamine. But the idea, in my opinion, is not really to induce supraphysiological levels of plasma glutamine. Other approaches, besides limiting the dosage of glutamine and taking the glutamine in smaller dosages with meals, would be to use arginine simultaneously, as I've discussed in past postings. The first article I linked to (together with other articles) suggest to me that the ratio of arginine to glutamine, in approaches to supplementation, would be an important factor in limiting some of these effects. But, for this and other reasons, in my opinion, the dosage of glutamine at which this type of effect might occur would be different for every person and would depend on his or her activity level. It's always good for a person to talk to his or her doctor about this type of issue. As with anything, there tends to be a therapeutic dosage range and a range at which side effects begin to occur. It's interesting that Spolarics et al. (1991) [Spolarics et al., 1991: (http://www.ncbi.nlm.nih.gov/pubmed/1872392)] found evidence that glutamine, along with fatty acids, is a major energy source for endothelial cells in blood vessels perfusing the liver. Free fatty acids (FFAs), similarly, can be major energy sources for cells in some tissues, under some circumstances, but also can inhibit mitochondrial functioning and interfere with insulin sensitivity and cause other problems, etc. The roles of glutamine in cellular energy metabolism aren't as murky as the roles of FFAs are, but the point is that physiological substrates and compounds tend to not simply be either "good" or "bad."

No, Wait...(Note on Tetrahydrobiopterin)

No, I guess they're saying, in those two articles that I cited in my previous posting, that tetrahydrobiopterin (BH4) can be reduced by either/both methylenetetrahydrofolate reductase and dihydrofolate reductase. I can't bring myself to read through them really carefully right now. There's lots of research showing that methylfolate or folate can influence the cellular redox state and the ratio of oxidized to reduced glutathione, and that could influence BH4 regeneration more indirectly than the actions of those folate-derived-cofactor-dependent enzymes may.

I'm not saying there isn't an effect of reduced folates on BH4 regeneration, but it's not clear to me that those effects would occur because BH4 would be more efficiently regenerated, merely because of the presence of an excess of reduced folates or merely by virtue of the capacity, in vitro, of DHF and methylenetetrahydrofolate reductases to reduce BH4. Vitamin C, for example, *can* reduce BH4, too, but vitamin C isn't likely, in my opinion, to produce effects that resemble those of methylfolate. The effects of methylfolate on endothelial nitric oxide synthase are really strong, and I'll try to find, at some point, this one article that shows those effects. Endothelial nitric oxide synthase (and not just tyrosine and tryptophan hydroxylases) also requires BH4 as a cofactor, and the BH4 regeneration/substitution concept has been suggested, by researchers, as a mechanism that would account for the vasodilatory effects of reduced folates. But I think that many of the effects of reduced folates can be explained in terms of the effects of folate-derived cofactors acting as substrates or allosteric activators/inhibitors of folate-cofactor-dependent enzymes.