Also, the italics on the "et al.'s" has been lost in pasting this. I tried to replace the lost formatting of Greek letters, such as the mu-M (shown here as uM) that refers to micromoles per liter, but I may have missed some of them. The "ug" or "uM" refer to micrograms or micromolar, respectively. I may have missed some, and the problem is that the Greek letter, "mu," shows up as an m, when the font is no longer in "symbol." I think I caught and replaced all of the "font-reverted" characters.
I also think that some of the studies showing enhanced effects of cell proliferation, in response to extracellular folate levels of 2-9 uM in the culture media, may be due to the transient effects that bypassing cobalamin-limited methionine synthase activity, with extremely large intracellular folate concentrations, can have on cell proliferation. I'm not suggesting that the serum or extracellular fluid folate concentration should be, in vivo, 2-9 uM. I just talk about it to stress the point that the pharmacology of folic acid has not been pinned down very thoroughly. The 5-7 mg/d range, of doses for folic acid, is probably around the upper limit of doses that are conservative, given that 35 mg/week (5 mg/d) maximizes the homocysteine reducing effect of folic acid. There are, as I talk about in this paper, homocysteine-independent effects, but the nature of those effects is not really well-understood. Some of it might have to do with the gluconeogenesis/glycolysis stuff I talked about in previous postings.
I only pasted one of the diagrams, and I'll post the rest in the next posting, here.
Effects of Folate on Purine and Pyrimidine Metabolism: Mechanisms in and Implications for Neurodegenerative Diseases
Abstract
Elevations in plasma homocysteine (Hcy) have been associated with neurodegeneration, and supplementation with cofactors, such as folic acid and vitamin B12, for one-carbon metabolism has been shown to effectively lower Hcy. Research has increasingly shown, however, that higher doses of folic acid produce metabolic effects that become independent of changes in Hcy disposal. Folic acid-derived coenzymes are cofactors for cytosolic serine hydroxymethyltransferase (cSHMT) and thymidylate synthase (TS), which, along with dihydrofolate reductase, comprise the so-called thymidylate cycle. TS is required for de novo thymidine formation, which is itself required for normal DNA synthesis and turnover and for the prevention of the deoxyuridine accumulation that produces uracil misincorporation, a form of DNA damage. Folate-derived coenzymes are also required for the de novo formation of inosine monophosphate, from which all other purines are derived. It is perhaps more noteworthy, however, that low folate levels also interfere with purine salvage, apparently by complex mechanisms that are poorly understood. The capacity of folate supplementation to normalize purine salvage, evidently by reducing the levels of 5-phosphoribosyl-a-pyrophosphate (PRPP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that are elevated under low folate conditions, may be an important mechanism, along with the limitation of uracil misincorporation, by which folate supplementation could protect against neurodegeneration. An examination of these effects, particularly in terms of folate and vitamin B12 dose responses, on nucleotide metabolism is likely to be important for understanding Hcy-independent effects, as in neuroprotection and protection of the vascular endothelium, of supplementation with folate and other cofactors. Purines and pyrimidines themselves have been shown to exert potent neuroprotective and neurotrophic effects on neurons and other cell types in the central nervous system, and the interactions of one-carbon metabolism with purine and pyrimidine metabolism, in general, will be discussed. Finally, various pharmacological and dosage considerations for folic acid and other one-carbon cofactors will be discussed.
Introduction
A deficiency of either folate or vitamin B12 (cobalamin) has, for a long time, been known to cause megaloblastic anemia, and cobalamin deficiency, in particular, has been known to produce severe and sometimes irreversible neurological damage (Carmel, 1995). In recent years, however, researchers have come to appreciate that even mild, or subclinical, cobalamin deficiency can also cause cognitive impairment, dementia, and neurological symptoms, such as peripheral neuropathy (Carmel et al., 1995; Carmel, 1996; Karnaze and Carmel, 1990). Additionally, scientists have expressed interest in the use of folic acid (Fournier et al., 2002) or methylcobalamin (Mayer et al., 1996) in the treatment of circadian rhythm abnormalities, such as the phase delayed syndrome of "sundowning" in patients with dementia. It is interesting that melatonin biosynthesis depends on the activity of hydroxyindole-O-methyltransferase (HIOMT), the enzyme that catalyzes the final step in the biosynthesis of melatonin (Claustrat et al., 2005). The activity of this enzyme would be expected to increase in response to the provision of adequate folate and cobalamin to pinealocytes.
It has been generally accepted that the megaloblastic, or macrocytic, anemia that results from either cobalamin or folate deficiency is largely the result of a decrease in the de novo biosynthesis of thymidine monophosphate (dTMP) in red blood cell precursors (Koury et al., 2000). This is because the de novo formation of dTMP from dUMP, by thymidylate synthase, requires adequate cytoplasmic 5,10-methylenetetrahydrofolate (5,10-CH2-THF), and the formation and regeneration of cytoplasmic 5,10-CH2-THF requires both adequate folate and cobalamin (Koury et al., 2000). When thymidylate synthase activity is not adequate, the ratio of dUMP/dTMP increases, producing DNA damage due to uracil (deoxyuridine) misincorporation into DNA (Koury et al., 2000). As a result, erythrocyte precursor cells undergo apoptosis or megaloblastic transformation, and these changes produce anemia.
Deficiencies of folate and cobalamin also produce elevations in homocysteine (Hcy), an amino acid whose remethylation into methionine is dependent on adequate folate- and cobalamin-derived coenzymes (Gregory et al., 2000; Herbig et al., 2002). A common assumption has been that folate and cobalamin supplementation exert their protective effects, particularly with respect to endothelial cells, by reducing levels of serum total homocysteine (tHcy) (Moat et al., 2004). According to this explanation, Hcy would work primarily by producing direct toxic effects (Riksen et al., 2003) on endothelial cells. Researchers have often found an elevated concentration of tHcy to be associated with a higher incidence of various neurological and vascular disorders or events, events that include ischemic stroke (Sachdev, 2005). Elevations in tHcy have sometimes been associated with impairments in cognitive functioning (McCaddon et al., 1998; Sachdev, 2005) or with increases in the risk of developing AD (Seshadri et al., 2002), but other researchers have failed to find similar associations (Ariogul et al., 2004). Additionally, the reduction of tHcy has not always produced robust improvements in cognitive impairment or reductions in the risk of stroke (Toole et al., 2004). Some of the shortcomings of homocysteine-reduction strategies could be explained in terms of inadequate dosages, such as of folic acid and other cofactors required for homocysteine metabolism, or metabolic heterogeneity among individuals in experimental groups. However, the tendency has been to view homocysteine metabolism as occurring independently of other pathways or processes that are influenced by, for example, an increase in the intracellular folate concentration.
Researchers have increasingly recognized that unbound, reduced Hcy is almost certain to be at too low a concentration to mediate the adverse effects associated with hyperhomocysteinemia (Riksen et al., 2003) and that other disturbances in one-carbon metabolism, apart from directly toxic effects of Hcy, warrant investigation. For example, hyperhomocysteinemia increases the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy), sometimes referred to as the transmethylation ratio (AdoMet/AdoHcy), and intensifies the inwardly directed gradient of adenosine (Ado), thereby increasing the intracellular AdoHcy levels and decreasing extracellular Ado levels (Riksen et al., 2003). Increases in AdoHcy levels have been shown to intensify TNF-a-mediated cytotoxicity, and a decrease in extracellular A2a adenosine receptor activation is known to be pro-inflammatory (Riksen et al., 2003). Changes in tHcy can also produce disturbances in the so-called "network" of folate-dependent enzymes involved in one-carbon transfer reactions, and these "network disruptions" (Field et al., 2006) can influence many other metabolic pathways. Changes in these metabolic pathways and physiological processes often accompany changes in tHcy and may explain some of the variability in cognitive functioning, and in other disease processes, among individuals that share a common tHcy level. A better understanding of the interrelationships of Hcy metabolism and other physiological processes may allow the benefits of Hcy reduction strategies to be augmented. In this paper, an attempt will be made to explore the mechanisms by which changes in homocysteine metabolism, and more broadly one-carbon metabolism, interact with other processes to disrupt nucleotide metabolism and impair endothelial and neurological functions. Uridine prodrugs have been found to exert neuroprotective effects against mitochondrial insults (Cansev, 2006; Saydoff et al., 2006; Wurtman et al., 2006), and there is reason to think that the neuroprotective effects of folate, and of other cofactors for one-carbon metabolism, are partially mediated by their effects on purine and pyrimidine metabolism. Inosine, which is hypoxanthine (HPX) riboside, has been shown to be neuroprotective (Di lorio et al., 2001; Haskó et al., 2004), by buffering ATP and adenine nucleotide levels and modulating glutamatergic transmission (Deutsch et al., 2005), and folate is required for de novo purine biosynthesis (van Ede et al., 2002). Although neurons in the adult brain depend very heavily on purine salvage (Deutsch et al., 2005), to maintain cellular purine pools, endothelial cells produce purines, by the de novo pathway, and provide them to cells in the CNS. Moreover, in non-mitotic tissues such as the liver, folate deficiency has been shown to produce a more pronounced inhibition of HPX salvage than, apparently, an inhibition of de novo HPX/IMP formation (Walzem et al., 1983).
Overview of Homocysteine and One-Carbon Metabolism
Homocysteine is generally metabolized by its remethylation to methionine, a process that is influenced strongly by folate and cobalamin status, and less strongly by the supplies of other cofactors, and by the transsulfuration pathway (Gregory et al., 2000; Herbig et al., 2002). The transsulfuration pathway tends to be activated when dietary protein and, more specifically, dietary methionine are abundant (Herbig et al., 2002). Dietary methionine is converted into AdoMet by either of two isoforms of methionine adenosyltransferase (MAT), and abundant AdoMet both activates the transsulfuration pathway and suppresses the remethylation pathway (Herbig et al., 2002; Santamaria et al., 2003). When dietary methionine is not abundant, the remethylation of Hcy serves to conserve and maintain the amount of available methionine (Herbig et al., 2002). The suppression or activation of either pathway is mediated, in part, by the allosteric effects of AdoMet on certain key enzymes. When abundant dietary methionine allows for abundant AdoMet biosynthesis, the allosteric inhibition of methylenetetrahydrofolate (5,10-CH2-THF) reductase (MTHFR) by AdoMet becomes metabolically significant and suppresses the remethylation pathway (Herbig et al., 2002). AdoMet also acts as an allosteric activator of cystathionine b-synthase, one of the enzymes of the transsulfuration pathway (Herbig et al., 2002). When dietary methionine is lacking and AdoMet levels are compromised, the dearth of AdoMet disinhibits the remethylation pathway and slows the transsulfuration pathway.
In addition to the classical dichotomy of remethylation and transsulfuration, the cytoplasmic and mitochondrial isoforms of serine hydroxymethyltransferase (cSHMT and mtSHMT) have emerged as pivotal regulators of one-carbon metabolism. First, the reaction catalyzed by each isoform is reversible and does reverse in vivo (Gregory et al., 2000). There is also evidence that cSHMT and mtSHMT commonly operate in opposite directions in a given cell (Gregory et al., 2000), thereby allowing for cycling of serine, glycine, and formate between the cytoplasm and the mitochondria. The cSHMT-mtSHMT cycle has increasingly been regarded as crucial to one-carbon metabolism, and serine has been regarded as an important one-carbon "donor" (Gregory et al., 2000). Broadly speaking, an increase in the cSHMT protein content, in the absence of a concomitant increase in the overall folate coenzyme pool, may tend to decrease MS activity, and thereby decrease homocysteine remethylation, and increase TS activity (Herbig et al., 2002). Although the issue of the "normal" directionality of cSHMT activity is an area of controversy, it is clear that the reversibility of cSHMT, in concert with the exchange of glycine and serine and formate between the cytosol and mitochondria, allows for one-carbon metabolism to be maintained in a complex and flexible manner when the overall folate pool is low. When the pool of folate coenzymes are abundant, such as would occur in the context of folate supplementation, maximizing the cSHMT protein content may help limit the inhibitory effect of very high concentrations of 5-formyl-THF and 5-methyl-THF (Fu et al., 2003) on cSHMT activity, thereby maintaining TS activity (Herbig et al., 2002) and limiting uracil misincorporation into mtDNA or nuclear DNA (Branda et al., 2002). Also, an increase in the provision and utilization of cellular iron (Oppenheim et al., 2000; Oppenheim et al., 2001) or zinc (Oppenheim et al., 2000) or in coenzymated vitamin B6 (pyridoxal 5’-phosphate, or PLP) will help to maximize cSHMT activity (Scheer et al., 2005) but will not necessarily reduce tHcy, if the intracellular folate pool is low. There are clearly numerous beneficial effects to be gained from maximizing folate status and minimizing tHcy, but a reduction in tHcy per se should not be viewed as the be-all and end-all of therapies related to one-carbon metabolism. Rather, Hcy reduction should be viewed as a first step and a general indication that the folate network is not being overtly "disrupted" (Field et al., 2006) by a relative lack of cofactor availability. Increases in the cellular iron pools and in PLP availability will tend to increase SHMT activity and limit uracil misincorporation but, by themselves, should not be expected to reduce tHcy.
Interactions of Folate With Uridine and Non-thymidine Pyrimidine Metabolism
Folate may also interact with the metabolism of pyrimidines in general ways, apart from merely allowing de novo thymidine formation to proceed normally, but these generalized pyrimidine effects are somewhat unclear and elusive. Methotrexate has been found to decrease both plasma hypoxanthine and plasma uridine (Smolenska et al., 1999) but increase the potentially cytostatic intracellular concentration of uridine 5’-triphosphate (UTP) (Fairbanks et al., 1999), and vitamin B12 deficiency was found to decrease the plasma uridine concentration (Parry and Blackmore, 1976a; Parry and Blackmore, 1976b) by almost 50 percent (Parry and Blackmore, 1976a), compared to B12-replete subjects. The mechanism for this effect of cobalamin deficiency is unclear (van der Weyden et al., 1979).
A more important series of interactions of folate and pyrimidine metabolism are likely to occur if uridine or uridine prodrugs are used therapeutically, such as in treating neurodegenerative disorders. In mice lacking the gene encoding CTP:phosphocholine cytidyltransferase-a (CT-a), the tHcy levels were found to be 20-40 percent higher than in wild-type controls (Jacobs et al., 2005). In contrast, the tHcy levels were reduced by about 50 percent in animals lacking the gene for phosphatidylethanolamine N-methyltransferase (PNMT) (Noga et al., 2003). The explanation for this is that both pathways produce phosphatidylcholine, and the activity of one pathway is upregulated when the other pathway is absent or reduced (Stead et al., 2006). The main point is that uridine could conceivably reduce tHcy levels by increasing CTP levels, thereby sparing AdoMet from being consumed by PEMT activity and reducing tHcy levels.
Maximizing thymidylate synthase activity, by maintaining intracellular folate levels, is also likely to be important if uridine or uridine prodrugs are used therapeutically in the long term. Mashiyama et al. (2004) found that supplementation of cultured cells with thymidine, deoxycytidine, and adenosine produced increased uracil misincorporation, and it was suggested that this effect was due to the conversion of excess deoxycytidine into deoxyuridine (Mashiyama et al., 2004). Although supplemental uridine would still have to be converted into deoxyuridine, by ribonucleotide reductase (Voet and Voet, 1995), it is possible that the uridine-induced expansion of the pyrimidine pool would make the protective effect of high intracellular folate levels, against uracil misincorporation (Mashiyama et al., 2004), more important than it otherwise would be. Thus, adequate folate supplementation may enhance the safety of the use of uridine or uridine prodrugs in neurodegenerative disorders.
Effects of Folate Status on Purine Metabolism
The de novo biosynthesis of hypoxanthine (HPX) is generally thought to be maintained fairly effectively under folate deficient conditions (Field et al., 2006), but recent findings about the cellular effects of the antifolate methotrexate (van Ede et al., 2002) suggest that folate status influences purine metabolism in more complex ways. First, it is relevant that some of the neuroprotective effects associated with higher serum folate may be the result of the disinhibition of de novo purine biosynthesis in endothelial cells, lining cerebral blood vessels, and of the activities of purine nucleoside salvage enzymes in the CNS. In the adult CNS, however, purine salvage is known to be especially important, in comparison to the contribution of the de novo pathway, for the maintenance of HPX and purine nucleotide pools (Pelled et al., 1999). For example, Pelled et al. (1999) noted that as astroglial cells mature, the contribution of purine salvage, over the de novo pathway, increases significantly.
It has become clear that the therapeutic effects of low-dose methotrexate are largely the result of increases in adenosine release, resulting from the interference by methotrexate with numerous enzymes involved in purine metabolism (van Ede et al., 2002). The increase in adenosine release appears to be an indirect result of the inhibition of GAR transformylase and AICAR transformylase activities, leading to the accumulation of AICAR and related compounds (see Figures 3 and 4). AICAR, or ZMP, a so-called "Z-nucleotide," exerts inhibitory effects on adenosine monophosphate deaminase by acting as a structural analog of adenosine monophosphate (AMP), and AICAriboside, the dephosphorylated form of AICAR, inhibits adenosine deaminase by acting as an adenosine analog (Foley et al., 1989). Thus, the inhibition of adenosine deaminase, HGPRT, and purine nucleoside phosphorylase, in response to prolonged methotrexate administration, could somehow result from AICAR accumulation or some other effect of methotrexate (van Ede et al., 2002). AICAR has also been shown to inhibit adenylosuccinate lyase (sAMP lyase), thereby causing the accumulation of sAMP (Foley et al., 1989), and methotrexate has also been shown to inhibit the amidophosphoribosyltransferase (APRT) enzyme that catalyzes the first so-called "committed" step in de novo purine biosynthesis (Sant et al., 1992). Polyglutamated MTX and polyglutamated dihydrofolates, which accumulate abnormally as a result of MTX-induced DHF reductase inhibition, appear to directly (i.e. noncompetitively) inhibit APRT (Sant et al., 1992).
AICAR and its metabolites have been shown to accumulate in cells treated with methotrexate, and folate deficiency has been shown to produce effects on purine metabolism that are similar to those of MTX. Interestingly, dietary folate deficiency produced significant reductions in HPX salvage into guanine and adenine (Walzem et al., 1983), which would result from a decrease in either hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or in the conversion of IMP, salvaged from HPX, into AMP or GMP or both. A decrease in the conversion of IMP into AMP or GMP would be expected to result from the inhibition, such as by AICAR, of either adenylosuccinate lyase or adenylosuccinate synthetase. The finding that folate deficiency decreased the adenine to guanine ratio (Walzem et al., 1983) could be viewed as evidence that the adenylosuccinate synthetase/sAMP lyase pathway was more significantly inhibited than the IMPDH/GMP synthetase pathway. A decrease in sAMP lyase activity is an attractive explanation for the reduction in HPX salvage induced by folate deficiency, given that AICAR inhibits sAMP lyase and that AICAR is known to accumulate, as indicated by increases in urinary AICAR excretion (reflecting cellular AICAR accumulation), in folate or cobalamin deficiencies (Walzem et al., 1983).
A more complex explanation of the changes in purine metabolism is necessary if the increases in PRPP, induced by folate depletion, are taken into account. For example, when the folate concentration supplied in the extracellular medium was decreased from 2.3 uM to 4 nM, the PRPP concentration increased by approximately 10 to 20 fold, in cell culture experiments across numerous cell types (Kane et al., 1987). Rennie et al. (1993) found increased PRPP levels in erythrocytes in folate-deficiency and noted that PRPP levels had been found to be inversely related to intracellular folate levels (Rennie et al., 1993). Additionally, Ghitis et al. (1987) found that PRPP levels were significantly elevated in folate-deficient monocytic cells or in the same cells treated with methotrexate. This is important for understanding the response, to low folate concentrations, of the de novo pathway for purine formation, because PRPP allosterically activates APRT (Voet and Voet, 1995). The elevations in PRPP levels, in response to low folate levels, are particularly important when viewed with the fact that folate deficiency has been shown to either not decrease (Walzem et al., 1983) or actually increase (Herbert et al., 1964) the activities of the overall, pre-AICAR-transformylase steps of the de novo pathway. In genetic HGPRT deficiency, for example, PRPP is known to accumulate and to be a major factor responsible for the resulting increase in the activity of the overall de novo purine biosynthetic pathway (Pelled et al., 1999) and, as a result, for the increase in AICAR levels (Sidi and Mitchell, 1985). Thus, the accumulation of PRPP due to low intracellular folate concentrations (Kane et al., 1987) would be expected to activate APRT, thereby leading to further AICAR accumulation (Walzem et al., 1983), and to be generally consistent with the impairment in purine salvage associated with folate deficiency (Walzem et al., 1983).
It is still unclear, however, what the primary mechanism is that results in PRPP accumulation. One possible scenario is that AICAR accumulation initially impairs purine salvage, that this leads to a decrease in the consumption of PRPP by HGPRT, and that the extra PRPP maintains or accelerates APRT and thereby exacerbates the accumulation of AICAR, producing a vicious cycle (see Figures 4 and 5). Barsotti et al. (2002) noted that PRPP formation depends indirectly upon purine nucleoside phosphorylase activity, which produces ribose-1-phosphate, but it is not clear how the inhibition of PNP activity, which has been shown to occur in cells of methotrexate-treated patients (van Ede et al., 2002), could result in PRPP accumulation. It is conceivable that AICAR directly inhibits HGPRT, by acting as an AMP analog, given that AMP and GMP inhibit HGPRT competitively (Murray, 1967). This inhibition of HGPRT could then account for the accumulation of PRPP due to low intracellular folate levels.
The mechanism for PRPP accumulation may be more complex, however, and result from more fundamental disturbances induced by folate deficiency or depletion. Rennie et al. (1993) noted that PRPP synthetase activity may somehow be elevated in folate deficiency (Hershko et al., 1969). Additionally, Kane et al. (1987) found some minimal evidence suggesting that the pentose monophosphate shunt may somehow produce more glucose-derived ribose in response to low folate levels and thereby lead to PRPP accumulation. AICAR also activates AMP-activated protein kinase (Rattan et al., 2005), and this action could somehow contribute to PRPP accumulation and to the other derangements of purine salvage that occur under low-folate conditions.
Finally, it is conceivable that the reduction in purine salvage under low folate conditions is partially the result of changes in the directionality of AdoHcy hydrolase. When tHcy is elevated, the extra Hcy exerts a mass action effect that drives the AdoHcy hydrolase reaction toward the formation of AdoHcy, rather than allowing the "forward" reaction to produce adenosine and homocysteine (Riksen et al., 2005). The net effect of this is to "initially" decrease intracellular adenosine but to then lead to an increase the import of extracellular adenosine, thereby decreasing extracellular adenosine and the activation of plasma membrane adenosine receptors (Riksen et al., 2005). This effect can become quite significant. For example, the elevation of tHcy from 6.7 uM to 14.7 uM caused the interstitial fluid, extracellular adenosine concentration in renal tissues to be reduced by about 50 percent (Chen et al., 2002). It is not clear how this inward shift of the transmembrane adenosine gradient would influence purine salvage, but it is possible that it somehow interacts with changes in PRPP or AICAR to derange purine salvage or de novo purine biosynthesis.
Contributions of Folate-Induced Thymidine and Hypoxanthine to Neuroprotection
There is growing evidence that the metabolic effects of folate on nucleotide metabolism, and not on Hcy reduction per se, can contribute to the cellular "protective" effects of folic acid on neuronal cells and other cell types. An important point is that both HPX and thymidine have been shown to be involved in DNA damage due to low folate levels. Thymidine and HPX, either alone or together, have been shown to rescue various cell types (Koury et al., 2000; Lin et al., 2006; Watkins et al., 1983), including cultured neurons (Kruman et al., 2002), from folate deficiency. Kruman et al. (2002) found that a combination of both 30 uM HPX and 15 uM thymidine significantly normalized the survival of folate-deficient and methionine-deficient cultured neurons. Folate and methionine deficiency was found to cause neuronal death in cultured neurons by inducing DNA damage, and Abeta1-42 significantly augmented the DNA damage caused in neurons made deficient in folate and methionine (Kruman et al., 2002).
It is also intriguing that DNA damage per se can consume large amounts of ATP and could be expected to exacerbate the "primary" abnormalities in nucleotide metabolism due to low folate levels. Kruman et al. (2000), for example, found that the ongoing repair of DNA damage can consume significant amounts of ATP and cause caspase-dependent cell death in neurons. Similarly, the apoptotic cell death that results from folate depletion can be merely the end result of a long "futile cycle" of attempted DNA repair or replication (Li et al., 2003). Thus, the "steady-state" degree of measurable DNA damage may not necessarily even capture the true metabolic cost due to folate depletion.
Many other studies have shown low folate levels to be associated with neurodegeneration, and this neuronal damage has frequently been shown to be related to increases in uracil misincorporation and other forms of DNA damage. For example, Snowdon et al. (2000) found that low levels of serum folate were strongly associated with higher levels of cortical atrophy found in participants in the "Nun Study." In mice, folate deficiency was found to more than double the infarct volume following experimental ischemia, and this increase in infarct size was associated with increases in DNA damage (Endres et al., 2005). Others have similarly noted that DNA damage induced by low folate levels or by methotrexate, DNA damage that includes uracil misincorporation and more severe forms of damage that result from uracil misincorporation, is likely to be involved in the resulting degeneration of neurons and other cell types (Blount et al., 1997; Goulian et al., 1980; Kruman et al., 2004).
The disinhibition of the forward reaction of AdoHcy hydrolase, such as occurs in response to lowering Hcy, may also favorably impact the processes leading up to Alzheimer’s disease. S-adenosylmethionine has been shown to reduce the amyloidogenic processing of amyloid precursor protein, by reducing presenilin-1 expression (Scarpa et al., 2003; Fuso et al., 2005). The mechanism for this effect is the normalization of the AdoMet pool that is available as a cofactor for DNA methyltransferases that maintain DNA methylation (Fuso et al., 2005). Lowering tHcy also disinhibits all methyltransferase reactions, by reducing the AdoHcy levels (Fuso et al., 2005). This is because AdoHcy inhibits most methyltransferase enzymes strongly (Fuso et al., 2005). It is unlikely that normalization of methyltransferase reactions can account for all the effects of folate on the brain or other tissues, however, given that the effects on, for example, endothelial cells become independent of AdoMet, AdoHcy, and Hcy levels as the extracellular folate level is increased into the micromolar range (Brown et al., 2006).
Given that AdoMet levels are unlikely to be increased in the central nervous system or in endothelial cells in a significant and concentration-dependent manner by a high level of serum folate (Brown et al., 2006), and given that folate-induced decreases in free Hcy, per se, are unlikely to account for folate-induced effects (Riksen et al., 2003), including vasculoprotection and neuroprotection, it follows that the folate-induced normalization of thymidine formation and purine metabolism are likely to play significant roles in the neuroprotective effects of folate. The notion that purine metabolism is mostly maintained in folate deficiency (Field et al., 2006) is simply not consistent with the decrease in purine salvage that occurs in folate deficiency (Walzem et al., 1983), the accumulation of PRPP that occurs in response to folate depletion (Kane et al., 1987; Rennie et al., 1993; Ghitis et al., 1987) or methotrexate administration (Ghitis et al., 1987) and that is likely to be due to a decrease in purine salvage, the methotrexate-induced impairments in purine salvage (van Ede et al., 2002), and the fact that both HPX and thymidine are required, sometimes individually but most frequently in combination, to rescue neuronal cells (Kruman et al., 2002) and other cell types (Koury et al., 2000; Lin et al., 2006; Watkins et al., 1983) from folate depletion or from methotrexate administration. It is reasonable to assume that ischemia under folate-depleted conditions, in particular, would unmask the derangements in purine metabolism and in thymidine metabolism and exacerbate the neuronal damage and vascular damage that result from ischemia. Numerous purine nucleotides have been shown to induce a multitude of neuroprotective effects (Di lorio et al., 2001; Haskó et al., 2004; Deutsch et al., 2005), and these nucleotide pools would be expected to be depleted in response to the folate-depletion-induced decrease in purine salvage (Walzem et al., 1983). Additionally, the improvements in vasodilation, in response to high-dose folate (Moat et al., 2004), are likely to be neuroprotective in and of themselves. The proposed mechanisms by which folate depletion would lead to neuronal damage are outlined in Figure 6.
Dosage and Pharmacological Considerations for Folic Acid and Other One-Carbon Cofactors
There is growing evidence that an expansion of the overall pool of cellular folate coenzymes, such as in cultured endothelial cells, can produce beneficial effects at folate concentrations higher than those required to minimize intracellular Hcy. For example, Brown et al. (2006) found that an increase in the concentration of folate improved the barrier function and differentiation of cultured endothelial cells without altering the AdoMet content or overall DNA methylation status. Similar effects could be produced by the addition of exogenous thymidine and hypoxanthine (Brown et al., 2006). Additionally, the endothelial cells appeared to be capable of storing fairly large "excess" concentrations of folate coenzymes (Brown et al., 2006). These findings are important for understanding the pharmacological aspects of folate supplementation. Although cSHMT is known to be inhibited by 5-formyl-THF and by 5-CH3-THF (Fu et al., 2003), for example, the in vivo significance of this inhibition is likely to vary in a cell-type-specific manner and may not become significant until the overall folate pool is greatly enlarged. There is almost no pharmacological data on the degree to which a given dosage of folic acid will or will not inhibit the activities of cSHMT or mtSHMT or other folate-dependent enzymes, but there is a need for this type of information.
For example, Brown et al. (2006) found that an increase in the culture medium from 23 nM folate, a level that is low but does not produce overt deficiency and cytostasis, to 9.3 uM produced pronounced improvements in the proliferation, barrier function, and cytoskeletal morphology of cultured endothelial cells. The intracellular concentrations of AdoMet, AdoHcy, and Hcy did not differ significantly in cells cultured in the two mediums, and the AdoMet/AdoHcy ratio and Hcy excretion rates also did not differ (Brown et al., 2006). These results clearly indicate that high folate concentrations produce "beneficial" effects that go beyond, and become independent, of Hcy reductions and AdoMet-dependent effects.
The potential for inhibition of cSHMT by 5-CHO-THF and 5-CH3-THF is a concern, but the effects of low micromolar (2.3-9.3 uM) concentrations of folate on cultured cells (Brown et al., 2006; Kane et al., 1987; Koury and Horne, 1994; Mashiyama et al., 2004; Wang et al., 2005) strongly suggest that cSHMT inhibition does not occur especially readily. It is unlikely that the concentration of intracellular total folates produced by 9.3 uM extracellular folate inhibited the activities of cSHMT and other one-carbon enzymes, because the inhibition of cSHMT and TS would have been expected to interfere with thymidine formation and cell division. The findings of a computational study (Nijhout et al., 2004) indicates that in mitotic cells, for example, the activities of DHF reductase and thymidylate synthase increase by 100-fold in response to progression through the cell cycle. In contrast, the activities of other enzymes in the folate network did not change by more than 7 percent (Nijhout et al., 2004). This essentially indicates that cSHMT is "rate-limiting" for the thymidylate cycle (see Figure 2) in rapidly proliferating cells. The enhanced mitosis in cultured endothelial cells in response to large increases in intracellular total folate (Brown et al., 2006) suggests that cSHMT, probably in concert with changes in directionality of mtSHMT, can nonetheless respond in a flexible manner and sustain the thymidylate cycle. That said, however, it may be necessary to maintain higher levels of intracellular cobalamin and PLP, to sustain cSHMT activity, when the intracellular total folate level is substantially increased, as discussed below.
As discussed previously, it appears that the remethylation of Hcy, and the reduction in tHcy, becomes maximal at doses of folate that are lower than those required for the maximal reduction of uracil misincorporation and for other folate-dependent effects. For example, Arnadottir et al. (2000) found that a dosage of 5 mg (5,000 ug) folic acid given three times per week produced a reduction in tHcy that was largely maximal. When the dosage was increased to 5 mg or 10 mg daily, the mean tHcy, across all patients, was not reduced significantly more (Arnadottir et al., 2000).
At relatively low extracellular folate levels, ranging from 12 to 25 nM, the intracellular total folate concentration will clearly be subsaturating and will produce metabolic responses, to changes in enzyme content or substrate availability, indicative of competition for folate coenzymes. The total concentration of folate-binding sites, which are determined by the concentrations of folate binding proteins and the numbers of binding sites per mole of binding protein, in the liver are estimated to be 5-10 times the intracellular concentration of total folate coenzymes (Anguera et al., 2006). The intracellular total folate level in the mammalian liver is assumed to be 25-35 uM, and the concentration of one folate-metabolizing and folate-binding enzyme, 10-formyl-THF dehydrogenase, is roughly 42 uM in the liver of the rabbit (Anguera et al., 2006). In the computational study by Nijhout et al. (2004), 20 uM was viewed as a normal intracellular total folate concentration. A number of studies, however, cast serious doubt on the assumption that the intracellular total folate concentration is normally 20 uM in humans. The intracellular total folate levels are very unlikely to be this high in extrahepatic cells, such as endothelial cells.
An examination and comparison of the effects of folate supplementation on rodents and humans suggests that standard dosages given to rodents may be significantly higher than dosages commonly given to humans and can be compared to human doses on a mg/kg body weight basis. These comparisons suggest that the intracellular folate concentrations in the hepatocytes and extrahepatic cells of the average human cannot be assumed to be 20-25 uM, and this is partly because the 25-35 uM range in rodent models is the result of a folate intake that is likely to be higher even than the intake of a human who is taking a "high-dose" folate supplement. Clarke et al. (2006) compared the effects of two doses of folate on plasma folate and on endothelial function in mice, and the doses given to mice were compared to human dosages on a mg/kg body weight basis. The lower dose of 5.7 ug/kg, given to mice, would indicate a 400 ug daily dose for a 70 kg human, and the higher dose, 71 ug/kg, given to the mice would be a 5,000 ug/70kg dose for a human. Although the argument might be made that a comparison of rodent and human intakes would be more appropriately made on the basis of mg/kcal diet, the responses of serum folate to various doses in mice (Kotsopoulos et al., 2005; Clarke et al., 2006) suggest that valid comparisons can be made on the basis of mg/kg body weight.
The relatively small and variable increases in serum folate in mice, in response to relatively high doses, are generally consistent with the changes in serum folate in humans given high-dose folic acid. Interestingly, the intracellular folate concentrations in the livers of rats given folic acid at the standard dose of 2 mg/kg diet for 22 weeks was roughly 19.75 uM (Kotsopoulos et al., 2005). The serum folate in rats given this dose was only approximately 53.6 ng/mL (121 nM) after 22 weeks, and four weeks of folic acid given at 8 mg/kg diet only produced a serum folate of 107.8 ng/mL (280.3 nM) (Kotsopoulos et al., 2005). The intracellular folate concentration in the mammary tissue of rats given the standard 2 mg/kg diet dose was only about 0.3773 uM, and it was suggested that even the 8 mg/kg diet dose may not have produced an adequate increase in the folate pool in the mammary tissue (Kotsopoulos et al., 2005). When these results are viewed in the context of the small and variable increases in serum folate found in other studies with mice (Clarke et al., 2006) and with humans, it is reasonable to suspect that the liver becomes preferentially saturated with total folate coenzymes and that the serum folate does not become very high until the folate binding sites in the liver have reached some degree of saturation. As will be discussed below, the RBC folate level also may increase "preferentially" and at the expense of (i.e. more rapidly than) the serum folate (which represents the "extracellular" compartment of, particularly, endothelial cells supplied with folate by the systemic circulation).
This general model would explain the remarkably small increases in serum folate that occur in humans given doses of 5-10 mg folic acid per day. Although one study found a mean plasma folate level of 310 ug/L (701 nM) following 6 weeks of 5 mg/day FA (Doshi et al., 2004), another study found that 4-8 weeks of 5 mg/day folic acid increased serum folate to only 40-41 nM (Liu et al., 2004).
Other studies have shown that 12 nM (Mashiyama et al., 2004) or 23 nM (Brown et al., 2006) or 100-400 nM (Koury and Horne, 1994) concentrations of extracellular folate produced minimal folate-dependent effects and could be construed as folate-deficient conditions. Mashiyama et al. (2004) found that a 12 nM concentration of extracellular folate produced essentially no effect, on cultured human lymphocytes, in limiting uracil misincorporation into the DNA. A concentration of 3,000 nM was required to minimize uracil misincorporation (Mashiyama et al., 2004). Koury and Horne (1994) found, similarly, that 0.01 uM (10 nM) or 0.2-0.4 uM (200-400 nM) folate produced essentially no reduction in the percentages of apoptotic, folate-deficient proerythroblasts. The percentage of apoptotic, folate-deficient proerythroblasts did not reach a minimum, at between 1.6 and 1.9 percent apoptotic cells, until the extracellular folate was increased to between 3 and 6.36 uM (3,000-6,360 nM) (Koury and Horne, 1994). This range is similar to the value of 3,000 nM required to largely minimize uracil misincorporation (Mashiyama et al., 2004) and the 9.3 uM concentration that substantially improved, in comparison to 23 nM, endothelial cell proliferation and morphology (Brown et al., 2006).
Mashiyama et al. (2004) noted that the lack of effect of 12 nM folate could suggest that the lower limit, which is 13.5 nM, of the normal range for serum folate in humans is not appropriate. Mashiyama et al. (2004) noted that the effects of 12 nM extracellular folate on cultured cells could be regarded as roughly, but not perfectly, comparable to the effects of 12 nM serum folate on endogenous cell types, which would include endothelial cells. A 12 nM concentration of serum folate would be somewhat more potent than 12 nM folic acid, as noted by Mashiyama et al. (2004), because serum folate is primarily 5-methyl-THF. Reduced folates, which include 5-methyl-THF, are able to be more efficiently transported into cells than the folic acid that was used in the lymphocyte cell culture medium (Mashiyama et al., 2004). Given the large differences between the 3,000-9,300 nM and 12-25 nM concentrations (a 250-750-fold difference) and the corresponding cellular effects of the different concentrations, the importance of the distinction between serum folate and extracellular folate in a culture medium is likely to be fairly small.
In other studies, Wang et al. (2005) found that 5,000 nM folate produced stronger antiinflammatory effects on cultured monocytes than 500 nM, and Beetstra et al. (2005) found that 200 nM folate was more protective against the g-irradiation-induced formation of micronucleated and binucleated lymphoid cells. The effect of a 20 nM folate concentration in cultured cells was viewed as comparable to the effect of 20 nM serum folate, and the authors noted that a normal serum folate level of 20 nM has previously been shown to be inadequate for protection against DNA damage (Beetstra et al., 2005).
Notwithstanding the effects of low micromolar concentrations of folate on cultured cells, the daily dose of folic acid that will produce 3 uM or 6-9 uM serum folate is not clearly known. In one study, in which the assay limit was enlarged to test for serum folate levels in the micromolar range, 15-60 mg of folate per day for four weeks was found to produce serum folate levels ranging from roughly 2.9 to 9 uM (Sunder-Plassmann et al., 2000). After four weeks, the serum folate levels were 4.7 mM (4,696 nM) after 30 mg/day, and 8,950 nM after 60 mg/day. Numerous studies have found that relatively high doses of folate, in the range of 5-10 mg/day, improve flow-mediated vasodilation but do not reduce Hcy more significantly than lower doses of folate do (Moat et al., 2004 and references therein). These studies are consistent with the finding that 9,300 nM folate did not reduce intracellular Hcy and AdoHcy more than 23 nM folate did, despite the fact that 9,300 nM folate produced significantly greater improvements in endothelial cell proliferation and morphology than 23 nM (Brown et al., 2006). The Hcy-independent effects of high-dose folate on endothelial functioning has been attributed to the action of 5-Me-THF as a tetrahydrobiopterin analogue or as a stabilizer of tetrahydrobiopterin (Moat et al., 2004), and 5-15 mg doses of folate have been viewed as supraphysiological. The results of Brown et al. (2006) imply, however, that these high doses of folate may in fact be "physiological" and may improve endothelial cell function by producing greater saturation of the folate coenzyme pool in endothelial cells with folate coenzymes.
The increases in serum folate in response to daily doses of 5-10 mg are remarkably inconsistent, but the inconsistency might be explained in terms of the preferential saturation of RBC folate. For example, Woo et al. (2002) found that 10 mg/day folic acid, given for one year, only increased the mean serum folate from 24 nM to 40 nM. There is clearly a shortage of studies in which increases in RBC folate and serum folate levels have both been measured, at different times in response to different dosages of folic acid. The large number of studies showing that high doses of folic acid, such as 10 mg/day, produce very marginal increases in serum folate could suggest that RBC preferentially accumulate folate, intracellularly, and do so at the expense of other cell types (such as endothelial cells).
If this general, conceptual "model" has validity, the serum folate would be expected to increase very slowly and relatively linearly as the folic acid dosage is increased up to about 2.5-5 mg per day (the dosage required to saturate RBC folate levels). Although this RBC-saturating dose of folic acid could be in the range of 10 mg/day in some individuals, the serum folate might be expected to increase more sharply in response to dosages exceeding this supposed "saturation dose." Peña et al. (2004) found that 5 mg/day of FA, given for 8 weeks, increased serum folate by only 14 nM, from 29 to 39.6 nM and 28 to 41 nM in each of two groups, but increased RBC folate by a mean of 467.2 nM (from 855-1410 nM and 911-1419 nM in each of two groups). Moens et al. (2007) found that 10 mg/day of folate increased RBC folate from 384.3 to 1182 ng/mL (869 nM to 2,671 nM) in one group and from 532.2 to 1208 ng/mL (1,202 nM to 2,730 nM) in a second group of patients. Doshi et al. (2004) found that 5 mg/day FA, given for six weeks, increased serum folate to a much larger extent, from 9.1 mg/L to 310 mg/L (21 nM to 701 nM), but most studies have shown much smaller increases in serum folate in response to the same dose. If the results of the cell culture experiments, discussed above, are any indication, even 701 nM can scarcely be regarded as a serum folate concentration that would produce saturation, or even anything approaching saturation, of intracellular folate levels in endothelial cells and other extrahepatic cell groups.
In conclusion, doses of folic acid ranging from 5-15 mg/day should be considered physiological and, when used in conjunction with adequate methylcobalamin, unlikely to cause problems. The potential need for higher levels of serum cobalamin, to ensure that the folate cycle is not inhibited in endothelial cells or other target cells when the serum folate level becomes elevated by high-dose folate, is an issue that remains to be explored and quantified.
Dosage Considerations for Vitamin B12 and Vitamin B6
If high-dose folate is to be used as a neuroprotective or vasculoprotective strategy, it is very important that adequate intracellular vitamin B12 (cobalamin) is present in conjunction with the elevated intracellular folate levels. Because the MTHFR reaction that produces 5-CH3-THF is irreversible, and adequate methylcobalamin is required for methionine synthase activity to "consume" 5-CH3-THF at a maximal rate, poor cobalamin status causes 5-CH3-THF to accumulate abnormally (Smulders et al., 2006). In this "methyl trap" hypothesis of cobalamin deficiency, the accumulation of 5-CH3-THF inhibits cSHMT and other enzymes in the folate network. Consistent with this, the inactivation of cobalamin, bound to methionine synthase, with N2O was found to cause the accumulation of 5-CH3-THF and to almost completely deplete THF from the livers of rats, effects that are consistent with inhibition of methionine synthase activity (Home et al., 2003). Smulders et al. (2006) found that 5-CH3-THF was relatively more abundant in the RBC’s of a patient with cobalamin deficiency, a finding that is also consistent with the methyl trap hypothesis of cobalamin deficiency. Home et al. (2003) did not find 5,10-CH2-THF to be depleted, a fact that implies cSHMT was providing adequate amounts of 5,10-CH2-THF to thymidylate synthase and that could even indicate that thymidylate synthase activity was being maintained. This is because the absence of THF, in response to cobalamin inactivation, could indicate that THF was in fact being produced by the coupling of TS and DHFR and then "immediately" converted back into 5,10-CH2-THF (see Figure 2). This should be viewed with caution, however, given that other studies have shown cobalamin deficiency to produce considerable uracil misincorporation (Choi et al., 2004), implying that, perhaps due to inhibition of cSHMT by 5-CH3-THF, the thymidylate cycle was not being maintained.
Moreover, as the total intracellular folate concentration increases, the importance of maximizing methionine synthase activity would be expected to increase as well. Thus, at high intracellular total folate concentrations, it is likely to become an issue of the absolute concentration of 5-CH3-THF, as a potential inhibitor of cSHMT, and not simply an issue of an increase in the proportion (as is implied on a most basic level by the methyl trap hypothesis) of total folates that exist as 5-CH3-THF.
While intravenous methylcobalamin has clearly been shown to dramatically enhance the Hcy-lowering effects of folic acid (Koyama et al., 2002), there is less information on the dosing of oral methylcobalamin. Typically, 1-2 mg has been used in conjunction with folate, but it is not known if achieving higher serum cobalamin levels will be beneficial or necessary to maintain normal folate metabolism under conditions of high (i.e. 1-2 uM or higher) serum folate. There is reason to think that higher doses of methylcobalamin, such as in the range of 2-5 mg per day, will be necessary or beneficial when the intracellular folate levels are increased. This is because the intracellular methylcobalamin levels in the livers of "control" rats, rats that had been fed "adequate" or presumably "standard" levels of cobalamin, was estimated to be 0.0082 uM (8.2 nM) (Yamada et al., 2000). It was noted that the Km of the apoenzyme of methionine synthase for methylcobalamin is 0.34 uM (340 nM) (Yamada et al., 2000). Thus, the intracellular methylcobalamin concentration, under conditions of "normal" cobalamin status, may be as little as 2.4 percent of the Km value for the binding of methylcobalamin to methionine synthase.
It is clear that there is a need for true dose-response data on methylcobalamin. For example, what intracellular methylcobalamin levels result from graded increases in the extracellular methylcobalamin concentration in a cell culture medium? To what extent is a higher intracellular methylcobalamin level necessary to prevent 5-CH3-THF accumulation, and other folate network disruptions, as the intracellular total folate level is increased greatly? These are potentially very important questions to be answered, if high-dose folate or L-methylfolate are to be used, in as safe a manner as possible, in the long term.
It is also important to emphasize that higher doses of oral cobalamin (methylcobalamin or hydroxocobalamin in particular) will be expected lead to higher serum levels of cobalamin. This is because about 1 percent of a dose of cobalamin is absorbed by an intrinsic-factor-independent pathway (Nyholm et al., 2003). The mechanism for this absorpotion is unknown, but it occurs reliably across a wide range of doses (Nyholm et al., 2003).
It is also noteworthy that, especially if higher doses are used, methylcobalamin is likely to be a preferable form in which to administer cobalamin. The amount of cyanide liberated from cyanocobalamin has been viewed with concern in heavy tobacco users (Freeman, 1999), in whom the cyanide burden is known to already be increased. As early as 1970 (Foulds et al., 1970), and on a number of occasions in the intervening years (Freeman et al., 1978; Freeman, 1992; Freeman, 1999), it was suggested that cyanocobalamin should be withdrawn from the market for these and other reasons (Freeman, 1992; Freeman, 1999). In a patient with a genetic disorder affecting cobalamin metabolism, cyanocobalamin, but not hydroxocobalamin, was found to produce respiratory depression, an effect that would be consistent with the actions of cyanide in an already-vulnerable individual (Harding et al., 1997). Although a dose of 1 mg of cyanocobalamin is almost certainly "safe" and effective for treating overt cobalamin deficiency, this may not be the case at higher doses. Finally, cyanocobalamin has been shown to exhibit inferior bioavailability in comparison to hydroxocobalamin or methylcobalamin (Hoffer et al., 2005), and methylcobalamin was found to be superior to cyanocobalamin for treating circadian rhythm abnormalities (Mayer et al., 1996).
Although the dosage range for methylcobalamin is theoretically very large, there is reason to be concerned about the use of very high doses. Some studies have used doses that are "high" but are more likely to be physiological, such as 3 mg (Mayer et al., 1996) or even doses as high as 6 mg (Moriyama et al., 1987). Kira et al. (1994) used 60 mg of oral methylcobalamin, which appears to be the highest dose of oral methylcobalamin ever used in a study. This is potentially problematic, in part because cobalamins can become tightly bound to albumin at high serum levels (Broderick et al., 2005), levels that exceed the levels at which the transcobalamin binding proteins become saturated. This is potentially problematic with respect to the kidneys, given that cobalamins are large, charged molecules. Although a toxicological study in rats found no obvious signs of toxicity at very high doses (Nava-Ocampo et al., 2005), the reabsorption of cobalamins by the proximal tubules is likely to be saturable (Nava-Ocampo et al., 2005). The saturability of reabsorption does not, however, necessarily preclude some sort of interaction of "reabsorbable" cobalamins, such as by way of their being cationic, with the proximal tubule cells. Similarly, and perhaps consistent with this, free cobalamin is known to accumulate in the kidneys during higher-dose supplementation (Birn et al., 2003). The kidneys have been regarded as a "storage organ" for cobalamin. Although cobalamin appears to be very low in cytotoxicity (Nava-Ocampo et al., 2005), and the saturability of reabsorption might be assumed to make excessive accumulation unlikely, very little is known about the use and effects of doses of methylcobalamin in the range of 10-60 mg/day.
The other main concern about very high doses, such as doses exceeding 5-6 mg per day, is that methylcobalamin and hydroxocobalamin can act as "reservoirs" of nitric oxide. Hydroxocobalamin and cobinamide, a cobalamin analogue, have been shown to be beneficial under conditions of sepsis (Broderick et al., 2006; Greenberg et al., 1995), and the forms of cobalamin, in these and similar situations, are thought to act as nitric oxide scavengers (Broderick et al., 2005; Broderick et al., 2006).
There is evidence, however, that hydroxocobalamin and other forms of cobalamin can act more like nitric oxide donors (Bauer, 1998), interacting reversibly with nitric oxide (Rochelle et al., 1995), and produce a prolonged, slow, ongoing action of nitric oxide (Colpaert and Lefebvre, 2000). This effect may, if nothing else, decrease the blood viscosity in an undesirable way or exert any number of other effects.
Although vitamin B6 (pyridoxine) supplementation has generally not been shown to lower Hcy levels reliably or significantly (Koyama et al., 2002), the vitamin B6-derived coenzyme PLP serves as a cofactor for SHMT. Three enzymes involved in the transsulfuration of Hcy require PLP as a cofactor (Scherer and Baker, 2000), but the enhancement of transsulfuration with supplemental pyridoxine, nonetheless, does not apparently reduce plasma Hcy significantly. As discussed above, vitamin B6 supplementation enhances SHMT activity in a linear manner, across a range of intakes (Scheer et al., 2005), and an increase in the intake of vitamin B6 was shown to enhance lymphocyte proliferation in women (Kwak et al., 2002). The rate of lymphocyte proliferation was positively correlated with plasma PLP, and the pyridoxine-dependent increase in lymphocyte proliferation was suggested to be potentially related to an increase in SHMT activity (Kwak et al., 2002). Additionally, the glycine contents in the livers of rats was inversely related to the dietary pyridoxine level, suggesting the involvement of PLP-induced SHMT activity (Scheer et al., 2005). Interestingly, the serum glycine concentration was strongly, and inversely, related to the supplemental level of folic acid in rats, and the glycine cleavage system did not appear to be responsible for the hyperglycinemia in folate deficiency (Dickson et al., 2005). These findings highlight the importance of SHMT activity to one-carbon metabolism in general. Thus, by enhancing cSHMT activity, pyridoxine supplementation may enhance the minimization of uracil misincorporation that is produced by moderate-to-high-dose folate supplementation. Doses of 100-200 mg per day of pyridoxine appear to nearly maximally increase the activity, and PLP saturation, of erythrocyte aspartate aminotransferase (Oshiro et al., 2005). Large intakes of pyridoxine have been shown to cause peripheral neuropathy, but this effect has almost without exception occurred as a result of long-term doses of 200-500 mg per day or higher [(1); McCarty, 2000]. This side effect of excess pyridoxine should be taken seriously, however, and long-term doses should essentially not exceed 150-200 mg/day.
Conclusions and Prospects for Future Research
In conclusion, there is considerable evidence to suggest that the neuroprotective effects of folate are due to increases in de novo thymidine formation and to the normalization of purine salvage processes. By ensuring an adequate supply of dTMP in neurons, an adequate level of intracellular folate will prevent an increase in the dUMP/dTMP ratio from producing uracil misincorporation into DNA and, as a result, ATP depletion and apoptosis. By ensuring an adequate pool of purines in either endothelial cells or neurons themselves, folate will allow the extracellular and intracellular neuroprotective and vasculoprotective effects of IMP and other purines to be maximized.
Although many of the studies examining the vasculoprotective effects of folate and one-carbon cofactors have produced considerable confusion, the use of folate and one-carbon cofactors has seldom been approached from a rigorous, systematic, pharmacological standpoint. There is abundant evidence that higher doses of folate are "physiological," but the dose response of target cell types, in vivo, to various doses of folate and methylcobalamin and pyridoxine has very rarely been examined. In the future, researchers could, for example, provide groups of animals with various doses of folic acid or L-methylfolate, at doses sufficient to raise serum folate to different levels, and then also provide either an abundant amount of methylcobalamin or a barely adequate dose. The same "two-dosage" approach could be used with pyridoxine. Following this approach, endothelial cells or other cell types could be analyzed for intracellular folate concentrations and for metabolic markers such as DNA damage or purine salvage. Although this would present challenges, there is a need for such systematic research.
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