Role of B Vitamins on the One-Carbon Transfer Pathways Flore Depeint A,B,∗, W

Chemico-Biological Interactions 163 (2006) 113–132

Mitochondrial function and toxicity: Role of B vitamins on the one-carbon transfer pathways Flore Depeint a,b,∗, W. Robert Bruce b, Nandita Shangari a, Rhea Mehta a, Peter J. O’Brien a a Department of Pharmaceutical Sciences, University of Toronto, Canada b Department of Nutritional Sciences, University of Toronto, Canada Available online 24 May 2006

Abstract The B vitamins are water-soluble vitamins that are required as coenzymes for reactions essential for cellular function. This review focuses on the essential role of vitamins in maintaining the one-carbon transfer cycles. Folate and choline are believed to be central methyl donors required for mitochondrial protein and nucleic acid synthesis through their active forms, 5-methyltetrahydrofolate and betaine, respectively. Cobalamin (B12) may assist methyltetrahydrofolate in the synthesis of methio- nine, a cysteine source for glutathione biosynthesis. Pyridoxal, pyridoxine and pyridoxamine (B6) seem to be involved in the regeneration of tetrahydrofolate into the active methyl-bearing form and in glutathione biosynthesis from homocysteine. Other roles of these vitamins that are relevant to mitochondrial functions will also be discussed. However these roles for B vita- mins in cell function are mostly theoretically based and still require verification at the cellular level. For instance it is still not known what B vitamins are depleted by xenobiotic toxins or which cellular targets, metabolic pathways or molecular toxic mechanisms are prevented by B vitamins. This review covers the current state of knowledge and suggests where this research field is heading so as to better understand the role vitamin Bs play in cellular function and intermediary metabolism as well as molecular, cellular and clinical consequences of vitamin deficiency. The current experimental and clinical evi- dence that supplementation alleviates deficiency symptoms as well as the effectiveness of vitamins as antioxidants will also be reviewed. © 2006 Published by Elsevier Ireland Ltd.

Keywords: Folate; Pyridoxal; Cobalamin; Choline; Mitochondrial toxicity; Methyl transfer

1. Introduction are required as cofactors or substrates for enzymes essential for cell function. One-carbon metabolism is Until the folate fortification program was imple- important for protein and DNA methylation and also mented in 1998, 25–50% of the US population was for the synthesis of nucleotides. The former reaction thought to be deficient in folate, while 5–10% of the requires first the transfer of a methyl group to S- population may have been deficient in vitamin B12 and adenosylhomocysteine (SAH), forming the universal 10% in vitamin B6 [1]. All three vitamins (Table 1) methyl donor S-adenosylmethionine (SAM), while the latter is dependent on one-carbon-bearing folate metabo- lites in thymidylate or purine synthesis. As both folates ∗ Corresponding author at: Leslie Dan Faculty of Pharmacy, 19 Rus- sell Street, Toronto, ON M5S 2S2, Canada. Tel.: +1 416 978 2716; and choline can provide the one-carbon group in the fax: +1 416 978 8511. reactions leading to SAM formation, the genetic risks E-mail address: fl[email protected] (F. Depeint). associated with DNA misincorporation or mutation may

0009-2797/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.cbi.2006.05.010 114 F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132

Table 1 Structures of B vitamins Type Name Chemical structure

B6 Pyridoxal

Pyridoxamine

Pyridoxine

B9 Folate

B12 Cobalamin

This table lists the major dietary forms of the three B vitamins reported in this review. be a greater risk for folate deficiency than that associated tion, metabolism and the essential role of the bioactive with protein or DNA hypomethylation. form of the molecules, concentrating on one-carbon For each of these water-soluble vitamins, we will transfer pathways and other vitamin-dependent reac- consider the biochemical evidence relating to absorp- tions relevant to mitochondrial functions. In another F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 115

Table 2 2. Folates Clinical disorders associated with vitamin deficiencies Vitamin Established condition Possible associations Folate was identified in the late 1930s as the nutri- of deficiency ent required to reduce anemia during pregnancy. Other Megaloblastic anemia Colon cancer possible folate associations have been described over the Neural tube defects, Cardiovascular disease following years and are listed in Table 2. Folate (B9) with perinatal Pediatric leukemia exposure Pediatric 2.1. Folate delivery from the diet to mitochondrial neuroblastoma enzymes Pernicious anemia Cardiovascular disease (macrocytic anemia Stroke Folate was originally identified in yeast and liver. The and subacute Impaired cognitive Cobalamin (B12) combined function vitamin is most abundant in unprocessed grains, oranges, degeneration of the Carcinogenesis dried beans, peas, eggs and green vegetables. Since spinal tracts with Osteoporosis 1998 processed grain products (flours, bread, cereals) polyneuritis) Neural tube defects have been fortified and have become a significant source Multiple sclerosis of dietary folate. Fortification of foods was associated Epileptic seizures Cardiovascular disease with a decrease in neural tube defects such as spina Anemia Colon cancer Pyridoxal (B6) bifida [5] and possibly pediatric neuroblastoma [6]. Renal failure Reduced cognition, Folate deficiency can result from an inadequate dietary depression Dermatitis Alzheimer’s disease folacin (folic acid) content, malnutrition or increased metabolic needs caused by pregnancy/metastatic cancer This table is a list of clinical consequences of deficiency of folate, or antifolate chemotherapy, e.g. methotrexate. cobalamin or pyridoxal. The links between the clinical symptoms and vitamin deficiency were based on clinical measurements of vitamin Folates in the foods are mostly bound to proteins levels in an affected population, regression of symptoms upon vitamin as polyglutamates that cannot be absorbed as such and supplementation, or animal models. Conditions that have been estab- must be hydrolyzed by proteases first to the monoglu- lished are listed on the left, and others that have been suggested are tamate forms before absorption in the small intestine. found on the right. These possible associations listed in the last column Absorption occurs primarily in the proximal jejunum have not yet been proven in a rigorous manner. via the reduced folate carrier of the brush-border. The carrier activity is maximal at pH 6.3, and is mediated issue in this issue, we described the role of other B partly through folate−/OH− system, and partly by vitamins in maintaining mitochondrial pathways of H+ co-transporters. Reduced monoglutamylfolate in energy production [2]. Vitamins B6 and B12, but not the portal blood is taken up by the reduced folate folate, play a role in those pathways, the consequences carrier in the liver sinusoidal membrane, mediated by of which will be discussed here. Current and potential H+ co-transport. Other cells may use a folate binding biomarkers of vitamin deficiency will be described, fol- protein (folate receptor) in the cell membrane and lowed by evidence for the therapeutic use of B vitamin mitochondrial membrane for the uptake of folate supplementation. Clinical conditions associated with and 5-methyltetrahydrofolate (CH3THF) [7]. Inside specific B vitamin deficiencies (proven or suggested) cells and mitochondria the folate monoglutamates are are listed in Table 2, based on data collected from Refs. elongated to the poly-␥-glutamate derivatives, catalyzed [3,4] and recent publications (not cited here). The aim by folyl poly-␥-glutamate synthetase [8]. This results of this review is to better understand the relationship in the retention and compartmentalization of folate between mitochondrial dysfunction caused by vitamin cofactors in the cell and the mitochondria. Fig. 1 shows deficiency and possible clinical outcomes. It is important the various metabolic fates of folate proposed for the to note that we are defining mitochondrial dysfunction cytosol and mitochondria fractions of the cell. In the as any direct or indirect change in the cellular functions hepatocyte CH3THF and homocysteine are converted that will affect reactions localized in the mitochondrial to tetrahydrofolate (THF) and methionine by a reaction matrix or that have consequences on mitochondrial catalyzed by methionine synthase (reaction 9, Fig. 1). stability (e.g. mitochondrial membrane potential). Folate is also reduced to dihydrofolate (DHF) and THF This will include pathways ranging from energy using NADPH, catalyzed by dihydrofolate reductase metabolism (reviewed in this issue [2]) but also the (reaction 1, Fig. 1) [9]. Tetrahydrofolate enters the antioxidant balance or more general mitochondria-based mitochondria and reacts with serine catalyzed by serine pathways. hydroxymethyl transferase (reaction 2, Fig. 1) to form 116 F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132

Fig. 1. Folate-dependent methyl-transfer pathways. Redrawn from Prasannan et al. (J. Biol. Chem. 278 (2003) 43178–43187) with a number of additions. Reactions are catalyzed by the following enzymes: (1), DHF reductase inhibited by methotrexate; (2), serine hydroxymethyltransferase; (3), 5,10-methylenetetrahydrofolate dehydrogenase; (4), 5,10-methenyltetrahydrofolate cyclohydrolase; (5), 10-formytetrahydrofolate synthetase; (6), methionyl-tRNA formyltransferase; (7), 10-formyltetrahydrofolate dehydrogenase; (8), methylenetetrahydrofolate reductase inhibited by flu- orouracil; (9), methionine synthase; (10), methenyltetrahydrofolate synthetase; (11), glycine cleavage system; (E), thymidylate synthase. Not all coenzymes are shown. Reactions 3–5 are catalyzed by tetrahydrofolate synthase. Methenyl-THF synthase (10) can also use leucovorin/folinic acid as substrate. It is important to notice the role of Vitamins B6 (reaction 2) and B12 (reaction 9) in methyl group transfer reactions. Other vitamins (mostly NADP+) are also involved at various levels of the pathways. SAM: S-adenosylmethionine; ADH: alcohol dehydrogenase; ALDH2: aldehyde dehydrogenase. glycine and 5,10-methylenetetrahydrofolate (CH2THF). and the survival of cells in culture. Thirty to fifty per- The latter is reduced to formate and THF, catalyzed cent of cellular folates are located in the mitochondria. by 10-formyltetrahydrofolate synthetase (reaction 5, It is generally thought that the mitochondrial folates are Fig. 1). The unique transporter identified in the mito- the primary actors of serine/glycine cycling, while the chondrial inner membrane that transports THF and other cytosolic folates are the primary location for thymidy- folates into the matrix has been named the mitochondrial late, purine and methionine synthesis. As shown in Fig. 1, folate transporter and has been sequenced [10]. the proper functioning of mammalian folate metabolism requires two separate independent compartments in the 2.2. Essential role of folate in mitochondrial and cell containing parallel but distinct folate metabolizing cellular function enzymes. The mitochondrial folate pool does not equi- librate with the cytosolic pool. Major folate metabolites Folates function as a family of cofactors that carry found in the cytosol were THF and CH3THF whereas in one-carbon (C1) units required for the synthesis of the mitochondria they were THF and formyltetrahydro- thymidylate, purines, and methionine, and required folate (CHOTHF). for other methylation reactions. Folate is essential for In the mitochondria reduced folates are required metabolic pathways involving cell growth, replication for three functions: (1) the initiation of mitochon- F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 117

Fig. 2. Choline metabolism and choline-dependent methyl-transfer pathways. The figure is compiled from various sources and represents the choline catabolism pathway. Reactions are catalyzed by the following enzymes: (1), choline dehydrogenase; (2), betaine aldehyde dehydrogenase; (3), betaine: homocysteine S-methyl transferase; (4), dimethylglycine dehydrogenase; (5), sarcosine dehydrogenase; (6), serine hydroxymethyl transferase; (7), glycine cleavage system; (8), phosphoethanolamine methyl transferase/phospholipid-N-methyl transferase (need three cycles of PEA to PC); (9), phosphocholine phosphatase. Reaction 6 is the only step of this pathway that is reversible. Choline itself can however be formed from phosphate ethanolamine or phosphatidylethanolamine through methyl transfer from SAM (8). A number of metabolites in this pathway interact with other pathways. For example betaine feeds into homocysteine metabolism (as an equivalent to methionine synthase depicted in Fig. 1), while glycine and serine are being used in a large number of reactions, including methyl transfer to or from tetrahydrofolate (Fig. 1, reaction 2) or formation of glutathione (Fig. 3). Again NAD+ plays an important role in the pathway, and reactions 4, 5 and 7 use FAD as cofactor while reactions 6 and 7 require PLP as coenzyme. drial protein synthesis; (2) glycine synthesis catalyzed In the cytosol the tetrahydrofolate metabolites carry by serine hydroxymethyl transferase; (3) serine cleav- C1 units in the form of formyl groups. These are used age to form 5,10-methylenetetrahydrofolate. Mitochon- for the synthesis of purines and thymidylate required for drial folate binding proteins include dimethylglycine cytosolic and mitochondrial ATP formation and DNA dehydrogenase and sarcosine dehydrogenase (reactions synthesis. As shown in Fig. 1,aCH3 group is transferred 4 and 5 Fig. 2) that catalyze the last two steps of to homocysteine from CH3THF via the formation of choline degradation [11]. Serine formed by cytoso- methylcobalamin intermediate. The reaction is catalyzed lic glycolysis enters the mitochondria and transfers a by methionine synthetase (reaction 9, Fig. 1) to form one-carbon group (CH2) to tetrahydrofolate, forming methionine. Methionine could then be used for cysteine glycine and CH2THF. The reaction is catalyzed by ser- synthesis required for GSH and protein synthesis as well ine hydroxymethyl transferase (reaction 2, Fig. 1, reac- as for the formation of SAM that serves as the one-carbon tion 6, Fig. 2), a pyridoxal phosphate (PLP)-requiring donor for the methylation of DNA, RNA, protein and enzyme. Mitochondria are thus the major synthetic site xenobiotics. Although liver mitochondria lack methio- for glycine and the one-carbon group donor CH2THF nine adenosyl transferase, SAM is readily transported in the cell. Although C1-substituted folates do not into mitochondria via a passive-carrier-mediated system leave the mitochondria, there is a two-way flow of [13]. C1 units between the mitochondria and cytosol in the In the formate pathway most of the methylating form of serine, glycine and formate for purine synthesis enzymes are reversible including the terminal step [12]. catalyzed by 10-formyltetrahydrofolate synthetase 118 F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132

(reaction 5, Fig. 1) that forms formate, ATP and tetrahy- tive stress [20]. Perinatal folate deficiency in mice also drofolate. The reverse of this step is a major detoxifying resulted in an increase in congenital defects including pathway for formate. Formate is converted to 10- neural tube defects with behavioral consequences [21]. formyl tetrahydrofolate catalyzed by the synthetase Increased plasma homocysteine levels are a risk factor and oxidation to CO2 catalyzed by 10-formyltetrahy- for cardiovascular disease, stroke and neurodegenera- drofolate dehydrogenase (reaction 7, Fig. 1) to reform tive disorders in humans. Homocysteine–thiolactone is tetrahydrofolate. a reactive plasma homocysteine metabolite formed from homocysteine by most cells as a consequence of the 2.3. Folate deficiency error-editing reactions of aminoacyl tRNA synthetases which ensure that homocysteine cannot complete the Folate status is commonly assessed as plasma or ery- protein biosynthetic pathway. Folate facilitates the con- throcyte folate. Deficiency can also be quantitated by an version of homocysteine to methionine and thus indi- increase in plasma levels of homocysteine (hyperhomo- rectly prevents thiolactone formation. Once the thio- cysteinemia) and glycine (hyperglycinemia), although lactone is formed it binds covalently to protein lysine homocysteinemia, as detailed later in this review, can residues. In the blood it is mostly bound to albumin also be induced by a number of other deficiencies and or hemoglobin. In human endothelial cells, however, it disorders. can induce a caspase independent apoptotic death that is Folate-dependent pathways and homocysteine pro- initiated by a collapse of the mitochondrial membrane vide a first approach to assessing deficiency. Folate potential [22]. It is not clear whether thiolactone forma- deficient diets in rats decreased serum folate and tion within cells reaches a high enough concentration to increased homocysteine [14], as well as cystathionine, produce the pathological changes associated with car- choline metabolism intermeditates (glycine, serine, N,N- diovascular or neutodegenerative disease. dimethylglycine, and N-methylglycine), but decreased tryptophan metabolites in NAD synthesis (kynurenine, 2.4. Prevention of oxidative stress and kinurenic acid, quinolinic acid) [15]. Folate and choline mitochondrial toxicity by folates deficiency decreased nicotinamide adenine dinucleotide (NAD) and NADP levels, even when niacin was present Folic acid can act as a direct antioxidant and scav- in the diet [16]. enger molecule. In vitro pulse radiolysis studies have Folate-dependent pathways and DNA damage pro- shown that folic acid can scavenge and repair peroxyl, vide still another class of markers. Folate deficiency hydroxyl and thiol radicals through oxidation of the increased reticulocyte micronuclei in mice and rats, purine hydroxyl groups [23]. Furthermore it can prevent suggesting that it resulted in chromosomal instability microsomal lipid peroxidation despite its water solubil- and chromosomal breaks [17]. In folate-methyl deficient ity [23].Invivo2-14C-folic acid is oxidatively cleaved at rats, lesion-containing hepatic DNA was less efficiently the C9–N10 bond to form p-acetamidobenzoic acid and methylated than lesion-free DNA and an increase in p-acetamidobenzoyl-l-glutamate which are excreted in DNA strand breaks preceded DNA hypomethylation. the urine [24]. The mechanism suggested is the oxi- This suggests that DNA lesions may be required for dation of the tetrahydrofolate by ROS to quinonoid the disruption of normal DNA methylation [18]. Folate dihydrofolate which undergoes an oxidative cleav- deficiency leads to chromosome break via uridine incor- age to yield 7,8-dihydropterin and p-aminobenzoyl-l- poration in DNA instead of thymidine [14]. During the glutamate. Ethanol or drugs increasing ROS increased DNA repair process induced by this misincorporation, these urinary products in hamsters. The increased folate single strand breaks are formed. If two uridine bases requirement by alcoholics could involve such a mech- have been introduced on the two DNA strands in close anism [24]. High dose folate supplementation also proximity, a double strand break that is more difficult decreased oxidative stress in cultured cells [25], but cyto- to repair can be formed and can lead to a chromosome toxicity was not evaluated. Homocysteine, when incu- break or sister chromatid exchange [19]. bated with aorta endothelial cells, produced oxidative Folate deficiency in the brain can be assessed in cell injury and lipid peroxidation, a result of mitochon- several additional ways. In mice folate deficiency, and drial toxicity (decreased respiration and RNA levels) increased homocysteine levels, decreased the number of induced by synergism between homocysteine and H2O2 proliferating cells in the hippocampus and induced DNA generated by autoxidation [26]. In hypertensive patients damage and apoptosis in rat hippocampal neurons in short-term supplementation with folic acid decreased association with mitochondrial dysfunction and oxida- oxidative stress biomarkers [27]. F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 119

Folate can prevent mitochondrial toxicity induced lished. The requirement for folate coenzymes for purine by methanol metabolites (Fig. 1). Methanol is oxidized and thymidylate synthesis has led to the use of antifo- to formaldehyde catalyzed by alcohol dehydrogenase late drugs e.g. methotrexate (inhibitor of dihydrofolate ADH1 which is then oxidized to formate catalyzed reductase (reaction 1, Fig. 1) and mitochondrial folate by mitochondrial aldehyde dehydrogenase ALDH2 or uptake) or fluorouracil (inhibitor of thymidylate synthase cytosolic GSH dependent formaldehyde dehydroge- (reaction E, Fig. 1)) in the treatment of cancer. These nase (ADH3). Formaldehyde and formate are inhibitors agents can inhibit tumor cell proliferation and induce of mitochondrial respiration [28]. Although research mitochondrial toxicity and apoptosis [36]. Methotrex- is required to substantiate this, formate formed in ate is still a drug of choice for treatment of childhood the cytosol and mitochondria is presumably detoxified leukemia in combination with citrovorum factor (leucov- by its respective 10-formyltetrahydrofolate synthetase orin). This combinational therapy allows very high doses and 10-formyltetrahydrofolate dehydrogenase activities of methotrexate to be used for optimal anticarcinogenic (enzymes 5 and 7, Fig. 1). Folate deficient patients might effect, followed by the antidote leucovorin to prevent thus be expected to be more susceptible to methanol or side effects and apoptosis of normal tissues. After more formalin. In fact, whilst the current antidote used in poi- than 40 years of use, the controversy over this use of son control centers for methanol poisoning is ethanol combination methotrexate therapy is still strong [37]. or an ADH inhibitor, folinic acid (leucovorin) therapy has recently been added to the American Academy of 2.5. Choline as an alternate methyl group donor Clinical Toxicology practice guidelines on the treatment of methanol poisoning [29]. Folate may also be useful Dietary choline was first shown to be important more against formaldehyde toxicity as its major metabolite, than 70 years ago. Choline prevented the development tetrahydrofolate, forms the condensation product 5,10- of fatty livers in rats fed diets with fat devoid of choline. methylenetetrahydrofolate (CH2THF) with formalde- Later it was shown that a deficiency of dietary choline in hyde [30]. animals resulted in liver cancer, and that such diets also Folate may affect riboflavin status. After folic acid promote breast, colon and other cancers [38]. Choline supplementation, riboflavin status was decreased in vol- was also found to be necessary for the growth of mam- unteers, while after a folate-enriched diet, riboflavin lev- malian cells in vitro. els were improved [31]. This was suggested as showing As shown in Fig. 2, choline is oxidized in the mito- that high folate intakes may increase the FAD require- chondria to betaine aldehyde, catalyzed by an uniden- ment for optimal activity of MTHFR, thus decreasing tified inner membrane oxidase (choline dehydroge- the circulating levels of FAD. nase (reaction 1, Fig. 2)), and then to betaine (N,N,N- Anti-folates have been important in cancer therapy trimethylglycine) catalyzed by NAD+ and betaine alde- and folate has been considered one of the more promis- hyde dehydrogenase (reaction 2, Fig. 2). Betaine is a ing chemopreventive agent. Individuals with the highest major mitochondrial methylating agent which main- folate intake have a 30–40% lower risk of develop- tains cellular replication, liver function and detoxifies ing colorectal cancer than those with a lower intake some phenolic xenobiotics. Betaine can also be used to of folate [32]. Folate deficiency increases colon car- decrease plasma homocysteine levels as betaine instead cinogenesis in mice and rats in some model systems of methyltetrahydrofolate can be used to re-synthesize [33] but decreases colon carcinogenesis in others (e.g. methionine from homocysteine, catalyzed by betaine: [34]). However in recent clinical trials supplementary homocysteine methyl transferase (reaction 3, Fig. 2) folic acid increased the frequency of recurrent colonic [39]. Choline metabolism by sequential demethylation polyps [35]. This ambiguity in the relationship between (reactions 4–7, Fig. 2) allows an alternative pathway folate deficiency and the risk of colon carcinogenesis for CH2THF regeneration from THF in cases where both in epidemiological studies and animal models was MTHFR activity is low. recently reviewed [32]. Folate is essential for the synthe- Choline can provide methyl groups for DNA synthe- sis, repair and methylation of DNA and folate deficien- sis. As the dietary requirement for choline is reduced cies increase their induction of mutations and result in by increased folate, methionine and vitamin B12, it has the induction of early steps of carcinogenesis. The vita- become customary to consider all these dietary com- min requirement increases at the site of the tumor due ponents together as sources of methyl donors. There to the higher rate of cellular divisions, and while folate appears, however, to be a specific requirement for dietary intake may prevent the early development of cancers, it choline, independent of the other methyl donors [40]. may also increase the growth of the tumor once estab- Current research on methyl group deficiency is largely 120 F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 focused on the effects of a deficiency of methyl donors on mitochondria and is thus a poor energetic substrate. In DNA methylation processes and on mutations resulting the mitochondria glycine readily methylates tetrahydro- from misincorporation of uracil in DNA. There are dra- folate with the aid of the mitochondrial glycine cleavage matic reductions in genome-wide methylation occurring enzyme system to form CH2THF, CO2 and NH3, which with proliferation [41] and gene-specific methylation has is detoxified by the urea cycle. Glycine is also converted been associated with carcinogenesis [42]. Mutational to serine for purine and phospholipids synthesis. events also occur, though they appear to primarily affect But glycine also plays roles unrelated to its carbon- mitochondrial DNA [39]. These effects, however, may transfer potential: (1) both serine and glycine are not explain liver carcinogenesis with choline-specific required as building blocks for the de-novo synthesis deficiency. of glutathione (GSH), an essential cellular antioxidant Recent studies suggest that liver failure of choline molecule (Fig. 3); (2) glycine is used in the cytosol for deficiency (steatohepatitis) is a consequence of reduced protein, sarcosine and creatine synthesis. Creatine and its phosphatidyl choline and reduced membrane integrity high-energy creatine phosphates act as a muscle energy [43]. Choline deficiency may increase oxidative stress store; (3) glycine provides nitrogen for the synthesis of in liver cells. It increases lipid oxidation in the nuclear other amino acids; (4) glycine is a building block in the fraction of liver cells [44] and 8-hydroxyguanine in DNA formation of aminolevulinate, an intermediate of heme [45], though it did not increase the malonyldialdehyde synthesis; (5) finally glycine forms glycine conjugates adduct [46]. Colon cancer can be induced in mice a diet (hippuric acids) with carboxylic acids catalyzed by N- with reduced choline, folate and methionine, and a sim- acyl transferase. ilar diet with reduced calcium and vitamin D3 [47] but Glycine availability in the cell is highly dependent this diet did not induce mutations in colonic cell DNA upon the one-carbon pathways. The reverse statement is [48]. The mechanism of carcinogenesis thus appears to also true. Folate and choline metabolism is dependent be quite complex. upon supplies of glycine/serine. Glycine may therefore play a role in a large number of essential pathways in 2.6. The central role of serine and glycine the mitochondria and the cell. In its other functions, glycine is central to the antioxidant defenses of the cell as Unlike other amino acids that are oxidized by the well as mitochondrial respiration and energy production TCA cycle, glycine is poorly oxidized by mammalian pathways. Therefore glycine or serine deficiency, due to

Fig. 3. Pyridoxal phosphate in homocysteine metabolism and glutathione biosynthesis. This figure is compiled from different sources. Reactions are catalyzed by the following enzymes: (1), cystathionine ␤-synthase (CBS); (2), ␥-cystathionase (GCT); (3), ␥-glutamyl cysteine synthase; (4), glutathione synthase. Although the pathway is localized to the cytosol, de novo synthesis of glutathione can have an impact on mitochondrial functions through its antioxidant capacity. Here again, a number of interactions between the different pathways and vitamins are evident. Serine and glycine from the choline catabolism pathway are both being used, but this time for their overall structure rather than methyl transfer properties. The first two reactions of the pathway are dependent upon PLP as a cofactor. Intermediate products of the reaction (alpha-ketoglutarate) also feed into another important mitochondrial pathway, the TCA cycle. F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 121 either folate or choline deficiency can lead to increased converted to the active coenzyme methylcobalamin via risk of oxidative stress to the cell. methionine synthase reductase, as part of the methionine synthase complex. It is not known how hydroxylcobal- 3. Cobalamin (vitamin B12) amin enters the mitochondria. In the mitochondria the cobaltic complex of hydroxycobalamin is reduced by Cobalamin was isolated and identified in the late reductase/GSH and is then converted by adenosyl trans- 1940s. Its potential as an antipernicious anemia factor, ferase and ATP to form cobaltous adenosylcobalamin however, had been recognized 20 years previously, with that can bind as a coenzyme to methylmalonyl-CoA the treatment of patients with fresh liver meat and liver mutase [53]. Cobalamins are released by the hepa- extracts. Other conditions associated with cobalamin tocyte into the plasma as methylcobalamin bound to deficiency are listed in Table 2. Only bacteria or microor- transcobalamin I or haptocorrin. This prevents loss by ganisms synthesize cobalamin and animals and plants do filtration through the kidneys but allows tissues to take not. The human need is very small, i.e. about 1 ␮g/day. up cobalamin via asialoglycoprotein receptors. While B12 deficiency has been shown to affect 10–15% of haptocorrin-bound cobalamin represents 80–90% of humans over the age of 60 in the US largely due to circulating form of vitamin B12, its role is unclear [54]. abnormalities in B12 uptake from the gut, resulting in Cellular uptake of cobalamin is mostly dependent on a functional B12 deficiency [49]. presentation with transcobalamin.

3.1. Cobalamin delivery from the diet to 3.2. Essential role of B12 in mitochondrial energy mitochondrial enzymes production and cellular function

Cobalamin is present in organ meats, milk and milk Cobalamin is involved in one-carbon transfer path- products, shellfish and other fish. There are three forms in ways as shown in Fig. 1. Methylcobalamin and CH3THF our diet. Adenosylcobalamin (coenzyme B12), methyl- act as cytosolic methyl carriers for methionine syn- cobalamin, and hydroxycobalamin are found in fish and thase which catalyses the conversion of homocysteine meat and particularly liver, whereas milk and cheeses to methionine (reaction 9, Fig. 1). Tetrahydrofolate pro- contains mostly hydroxycobalamin. Strict vegetarians duced in this reaction can receive a one-carbon unit as need to take cobalamin regularly as a supplement. CH2 from serine. The resulting CH2THF is required to Cobalamins are released from dietary proteins by synthesize deoxythymidine monophosphate (dTMP), a the acid environment and pepsin in the stomach. In both DNA precursor. The exact cause of megaloblastic ane- cases, the gastric and early small intestine pH can be mia associated with cobalamin deficiency is not known increased to levels that prevent dissociation or absorp- but it is likely a consequence of the impairment of DNA tion of cobalamin. The risk of cobalamin deficiency, synthesis rather than hemoglobin synthesis. It has also even with a cobalamin-rich diet, can thus be increased been suggested that the methionine synthetase inhibition in the ageing population [50] and for chronic users of in B12 deficiency causes an accumulation of methylte- antacid drugs [51]. Once released from dietary proteins, trahydrofolate that, in turn, results in a functional folate cobalamins bind to salivary R binder polypeptide. The deficiency and an inhibition of thymidylate synthetase. binder protein is digested in the small intestine by In the mitochondria adenosylcobalamin is required pancreatic trypsin and the cobalamins are transferred for the synthesis of succinyl-CoA from the carbon to stomach-derived intrinsic factor (IF) forming a atoms of valine, isoleucine, methionine or metabolites of IF/Cbl complex. This complex interacts on ileal mucosa odd-numbered fatty acids. The propionyl-CoA formed with IF/Cbl receptor, also identified as cubilin, and is converted to l-methylmalonyl-CoA which under- is transported into the enterocyte by endocytosis to a goes an adenosylcobalamin dependent isomerization to lysosomal vesicule [52]. In the lysosomes, the complex succinyl-CoA, an important intermediate of the TCA is dissociated by an unknown mechanism and cobal- cycle. Adenosylcobalamin is formed in the mitochon- amin, likely as hydroxycobalamin, crosses the basal drial matrix from reduced hydroxycobalamin and ATP membrane bound to newly synthesized transcobalamin catalyzed by adenosyl transferase. II into the portal blood [52]. In the hepatocyte, TC/Cbl complex is recognized by transcobalamin receptors and 3.3. Cobalamin deficiency leads to endocytosis into the lysosomes and endosomes where proteolytic digestion release hydroxycobalamin. Vitamin B12 status is commonly assessed by mean Hydroxylcobalamin is released in the cytosol and corpuscular volume or serum or plasma vitamin B12. Its 122 F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 status can be inferred from concentrations of methyl- ine [15], as well as decreased folate metabolism [63]. malonic acid (MMA), homocysteine, formiminoglu- In the rat, cobalamin deficiency decreased liver activity tamic acid, propionate, methylcitrate levels or holo- of l-serine dehydratase [63] with accumulation of the transcobalamine II. upstream choline metabolite, dimethylglycine [15]. B12 Dietary B12 deficiency is difficult to induce in ani- deficiency also increased plasma levels of serine, thre- mals. It requires long periods on special diets and yields onine, glycine, tyrosine, lysine, histidine [63], methyl- a heterogeneous metabolic defect. Although rats on a citric acid and propionyl-CoA [62] as well as increased cobalamin deficient diet for 24 weeks do not develop NAD+ to NADH ratio in the liver. megaloblastic anemia they do develop methylmalonic MMA accumulation is also hypothesized to affect aciduria and neuropathy [55]. Cobalamin deficient rats mitochondrial respiration. MMA was reported to inhibit will, however, develop megaloblastic anemia-like symp- complex II and ␤-hydroxybutyrate dehydrogenase toms if they are exposed to 10% oxygen for an additional [64,65]. After only 2 weeks of cobalamin deficiency 6 weeks so as to induce increased hematopoiesis [55]. (induced by cobalamin analogue), liver mitochondrial Cobalamin deficiency also affects folate metabolism. mRNA expression was reduced. Polycistronic mRNAs Rats on a cobalamin deficient diet have a methion- accumulated and by 5–6 weeks the activity of complex ine synthetase inhibition. The resulting hypomethylation III (cytochrome b) and IV (cytochrome a/a3) of the could explain the anomalies in the base substitution and electron transport chain was decreased [66]. Cobalamin decreased methylation of DNA [56] and could explain deficiency increased mitochondrial matrix enzymes the increased liver and colon carcinogenesis observed such as citrate synthase and succinate dehydrogenase. with B12 deficiency [57,58]. Cobalamin deficiency also It also inhibited the electron transport chain and state-3 leads to increased CH3THF and decreased CH2THF oxidation via glutamate, succinate, or duroquinol, and levels, resulting in increased dUMP production and mis- increased CoA and fatty acid synthesis [67]. The findings incorporation into DNA. Cobalamin deficiency inhibits linking cobalamin deficiency and mitochondrial respira- methionine synthase, thus leading to increased homo- tion are ambiguous, the authors suggesting that complex cysteine concentrations [19]. Cobalamin deficiency does II inhibition may be a consequence of the accumulation not affect free folate levels in patients but is associated of MMA metabolites methylcitric acid (MCA) and with decreased levels of polyglutamate folate, suggest- malonic acid (MA) rather than a response to MMA itself ing inhibition of synthesis of the folate coenzyme form. [68]. The exact nature of the inhibitor however does not The consequence is an inhibition of single unit carbon contradict earlier findings as all three metabolites are transfer required for DNA and RNA synthesis, glycine increased in methylmalonic aciduria [64]. More inves- to serine conversion, histidine breakdown and homocys- tigations are still needed to fully understand the physi- teine to methionine conversion [59]. ological impact of cobalamin deficiency on the electron Cobalamin-dependent metabolism has multiple transport chain and mitochondrial energy pathways. metabolic effects although only two enzymes have an absolute cobalamin coenzyme requirement. Inhibition 3.4. Prevention of oxidative stress and of methylmalonyl-CoA mutase (deoxyadenosylcobal- mitochondrial toxicity by cobalamin amin dependent enzyme) leads to increase of plasma MMA and increased urinary excretion [60]. The accu- There have been no reports that vitamin B12 pre- mulation of serum MMA is a reliable test of cobal- vents mitochondrial toxicity or has antioxidant proper- amin deficiency or hereditary methylmalonyl aciduria. ties, though B12 is known to be an efficient radical trap Methylmalonic acidemia and mitochondrial toxicity due [69]. Cobalamin deficiency however has been shown to to MMA excess lead to inhibition of carbamyl phosphate cause mitochondrial toxicity as a result of inhibition of synthetase I, pyruvate carboxylase and the dicarboxylate the TCA cycle [70]. In megaloblastic anemia red blood carrier required for the malate shuttle (reviewed in [61]). cell hemolysis is frequently high. There is frequently Defect in N-acetylglutamate synthase, glycine cleavage hyperhomocysteinemia, lipid peroxidation and evidence enzyme and pyruvate carboxylase can result in hyperam- of protein bound homocysteine in red blood cell ghosts, monemia, hyperglycinemia and hypoglycemia, respec- suggesting that cobalamin deficiency can result in oxida- tively. There can also be increased hepatic pyruvate tive stress-induced cell death from high intracellular oxidative metabolism and lactic acidosis [62]. Methion- concentrations of homocysteine [71]. Vitamin B12 can ine synthase is dependent on both CH3THF and cobal- enhance the effectiveness of folate to reduce high blood amin. Inhibition of this enzyme leads to increased levels homocysteine levels and oxidative stress. Indeed cobal- of homocysteine and metabolites such as cystathion- amin supplementation of patients with end stage renal F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 123 disease can decrease plasma homocysteine, MMA excre- tion and serum folate levels without having an effect on erythrocyte folate [72]. Currently there are more than 20 prospective, worldwide, interventional trials involv- ing at least 100,000 participants that are examining the effect of supplementary B-vitamins on the concentration of plasma homocysteine and morbidity and mortality from atherosclerotic vascular disease [73].

4. Pyridoxal (vitamin B6)

The term “vitamin B6” covers the three vitaminers: pyridoxal, pyridoxine and pyridoxamine (Table 1). Pyri- doxine, pyridoxal (PL) and pyridoxal phosphate (PLP) are the major dietary forms of the vitamin. Pyridox- ine was first identified in 1938. The catalytically active aldehyde (pyridoxal) or amine (pyridoxamine) and their phosphates were discovered in the early 1940s. Vitamin dependency was first recognized in 1954 with a reces- Fig. 4. Pyridoxal protein adducts. These are examples of protein sively inherited condition in which children develop adducts formed by pyridoxal phosphate. epileptic seizures beginning early in life [74]. Clini- cal evidence of vitamin B6 deficiency is not common believed that this B6 antagonist effect may be because because of its widespread occurrence in foods but Table 2 the drug reacts with PLP to form a Schiff base, which gives conditions associated with deficiency of the vita- depletes cytosolic B6 levels (and inactivates the drug). min. Approximately 10% of the US population con- Furthermore the Schiff base hydrazone products formed sumes less than half of the RDA for B6 and could be with hydrazine carbonyl reagents or drugs (e.g. iso- at an increased risk of cancer, neural decay and acceler- niazid, semicarbazide, hydrazine and hydroxylamine) ated aging [49]. compete with B6 for the B6 enzyme binding site and prevents the formation of the PLP coenzyme [77]. Other 4.1. Vitamin B6 delivery from the diet to mechanisms of pyridoxal kinase inhibition by drugs mitochondrial enzymes have been described [78]. The in vivo formation of a Schiff base of the antidiabetic drug aminoguanidine with Vitamin B6 is synthesized by plants and products pyridoxal as shown in Fig. 4 has also been described that are particularly rich in B6 are vegetables, whole [79]. The increase in tryptophan metabolism to xan- grain cereals, nuts, and muscle meats. An increased thurenic acid could be because B6 dependent enzymes the risk of B6 deficiency can be observed in advanced involved in the competing tryptophan metabolic path- age, severe malnutrition, excessive alcohol ingestion, way to anthranilic acid and nicotinic acid were inhibited. and in dialysis. Thermal losses of B6 in food process- Furthermore the peripheral neuropathy induced in some ing and preservation vary from 49% to 77% in canned patients by the chronic administration of isoniazid for foods or infant formula probably because any protein tuberculosis can be prevented by pyridoxine adminis- lysine present reacts with pyridoxal at 37 ◦C to form a tration. Similar results were obtained for phenelzine or Schiff base which rearranges to ␧-pyridoxylidenelysine hydralazine. Indeed excessive supplementation with B6 as shown in Fig. 4 [75,76]. can affect the efficacy of these drugs, e.g. levo-dopa, Drugs such as isoniazid, cycloserine, hydralazine and hydralazine, phenytoin [77]. penicillamine, or the anti-Parkinson drug levo-dopa can Vitamin B6 is absorbed from the diet mainly by also deplete tissue B6 levels. B6 deficiency can be simple diffusion across the enterocyte brush-border of assessed with the in vivo rat tryptophan loading test, the jejunum. Enterocyte and other cell plasma mem- which shows a marked increase tryptophan conversion branes are not permeable to PLP, and PLP needs to to xanthurenic acid which is reversed by the concomi- be dephosphorylated by intestinal phosphatases prior tant administration of pyridoxine. Pyridoxal kinase inhi- to being taken up by enterocytes [80]. The three B6 bition is also involved, and although the mechanisms vitamers are then phosphorylated (i.e. metabolically involved are not known and poorly understood, it is trapped) in the enterocyte but require dephosphorylation 124 F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 by plasma membrane phosphatases (similar to alkaline by phosphorylation metabolic trapping catalysed by the phophatases) before being released into the portal vein kinase (reaction 1, Fig. 5). Kinases first form pyridoxine- for transport to the liver. About 10% of the total body 5-phosphate and pyridoxamine-5-phosphate. These are pool of PLP is in the liver and about 20% of cellular B6 oxidised at the 4 position by a cytosolic pyridoxine phos- is located in the liver mitochondrial matrix as PLP. Pyri- phate oxidase (reaction 2, Fig. 5) that requires flavin doxine, pyridoxal and pyridoxamine are taken up rapidly mononucleotide (FMN) and O2. The PLP formed is by hepatocytes and as shown in Fig. 5 is accumulated then transferred to PLP-dependent apoenzymes [81,82].

Fig. 5. Metabolism of B6 vitaminers: metabolic conversion of B6 vitaminers catalyzed by: (1) pyridoxal (PL) kinase, (2) pyridoxal phosphate (PLP) oxidase, (3) B6 vitamin kinase conversion can be reversed by phosphatases, (4) any unbound pyridoxal is oxidised by aldehyde oxidase/aldehyde dehydrogenase 2 (ALDH2) to form pyridoxic acid which is released into the plasma and excreted in the urine. F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 125

The kinase is more active with PL than pyridoxine in is not known which of the B6 vitaminers added to cells humans and more active with PL than pyridoxamine is the best at preventing cytotoxicity induced by mito- in rats [82]. Pyridoxamine incubated with hepatocytes chondrial toxins. undergoes similar steps to form PLP with pyridoxamine phosphate oxidase activity [82]. 4.2. Essential role of vitamin B6 in mitochondrial Recently the 3D structure of pyridoxal kinase from and cellular function sheep brain and its complex with ATP has revealed that it is a dimmer and that pyridoxal binding causes the pro- Vitamin B6 is involved in aminotransferase reactions. tein to become more compact and the pyridoxal more Mitochondrial function is more dependent on PLP than tightly bound [83]. The kinase also forms complex with other subcellular organelles for a number of reasons. PLP the oxidase and transaminase that prevents the release of functions as a coenzyme for transaminases that partici- free PLP and hydrolysis by phosphatases. The 3D struc- pate in the catabolism of all amino acids by the urea cycle ture of the oxidase shows it is also a dimer containing an of the mitochondria. Accordingly the human require- essential arginine, required for binding of PLP. The oxi- ment for pyridoxine increases as protein intake increases. dase is inhibited by dicarbonyls. Mitochondria have no The chemical basis for PLP catalysis is to enable its kinase or pyridoxine/pyridoxamine-P oxidase activities carbonyl group to first form a Schiff base with the so pyridoxal phosphate is taken up by liver mitochon- amine component of the amino acid substrate. The pro- dria by passive diffusion facilitated by protein binding tonated form of the pyridoxal phosphate then acts as an which acts as a store [84]. Mitochondria may also take electrophilic catalyst, before the Schiff base is cleaved. up pyridoxamine-P by passive diffusion as mitochondria Transaminases are of two types in the way they utilize contain similar amounts (45% each) of pyridoxal-P and PLP: either forming ␣-oxo acids from ␣-amino acids, pyridoxamine-P whereas pyridoxine-P only accounts for or forming primary amines via its decarboxylase cat- 2% of the B6 vitaminers [85].[14C]pyridoxine incubated alytic action. Not all transaminases, however, are inhib- with hepatocytes forms mostly PLP, and releases both ited during pyridoxine deficiency. Transaminases also PL and PLP into the plasma [86]. Presumably PLP is participate in the malate aspartate shuttle that enables released as an albumin complex to the plasma. PLP also mitochondria to oxidize NADH formed by glycolysis. binds avidly to other plasma proteins such as transfer- Transaminases can also link amino acid metabolism to rin, or ␣-glycoproteins through formation of Schiff bases energy production through two reactions: first alanine (aldimine). Alternatively, the hepatocyte can release PLP can form pyruvate, feeding into the glycolysis pathway, as the albumin complex. and second glutamate can form ␣-ketoglutarate, feeding Any unbound pyridoxine or pyridoxal in the hep- directly into the TCA cycle. atocyte is oxidatively inactivated by aldehyde oxi- Vitamin B6 is also involved in decarboxylation reac- dase/aldehyde dehydrogenase 2 (ALDH2) to form 4- tions. Pyridoxal phosphate is a coenzyme for aminole- pyridoxic acid which is released into the plasma and is vulinate synthase located in the mitochondrial matrix, excreted in the urine [82]. Pyridoxic acid is mostly the which catalyses the initial and rate limiting step for only B6 vitamer found in the urine. The rate constants heme synthesis from glycine and succinyl-CoA. The for the various steps in Fig. 4 and proposed for rat and synthase requires rapid synthesis as it has a short half- human liver show that the kinases/oxidases (reactions 1 life (1 h) probably because the aminolevulinate product and 2, Fig. 5) are faster than the phosphatases (reaction can inactivate the synthase by reacting with the pyri- 3, Fig. 5) [82]. doxal carbonyl or can form ROS by autoxidation. Thus Plasma levels of B6 vitamers in order of concentra- a B6 deficiency could increase cellular endogenous ROS tion are PLP > pyridoxine > pyridoxic acid  pyridoxal formation because of a heme/cytochrome deficiency. For with much lower levels for pyridoxine-P, pyridoxamine- instance, a deficiency of mitochondrial complex IV could P or pyridoxamine [86]. By contrast pyridoxamine-P and impair respiration and accelerate the mitochondrial or PLP are the major B6 forms found in the tissues and the cell aging process [49,87]. mitochondria. The intracellular pathways of pyridoxam- Vitamin B6 is a cofactor for enzymes involved in side- ine have been much less studied and it has not yet been chain cleavage reactions and as such can play a role in a demonstrated that mitochondria take up pyridoxamine-P. large number of pathways: (1) PLP is a cofactor for two Presumably the pyridoxamine is not released from liver enzymes involved in cysteine formation, as detailed in cells as plasma pyridoxamine or pyridoxamine-P levels Fig. 3: the hemoprotein cystathionine ␤-synthase (CBS are much lower than the other B6 vitaminers. How PLP is (reaction 1, Fig. 3)) that removes serine from homocys- released by hepatocytes remains controversial. Finally it teine to form cystathionine, and ␥-cystathionase (GCT 126 F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132

(reaction 2, Fig. 3)), that then forms cysteine. Vita- assessed by erythrocyte tryptophan catabolites levels, min B6 is coenzyme of ␥-cystathionase. It releases ␣- aspartate aminotransferase and alanine aminotransferase ketoglutarate that can feed into the tricarboxylic acid activities, or plasma homocysteine. Pyridoxine defi- (TCA) cycle, increasing production of NADH later used ciency can also be diagnosed by increased urinary 4- for energy metabolism [2]. In this respect the action of xanthurenic acid, after a load test of 2 g oral tryptophan. vitamin B6 is similar to that of vitamin B12, which is Urinary cystathionine and plasma homocysteine levels a coenzyme of methylmalonyl-CoA mutase that leads are also measured after a test dose of 3 g methionine to the formation of succinyl-CoA that also feeds into [3]. the TCA cycle as noted above. Vitamin B6 deficiency By-products of pyridoxal-dependent reactions could does not decrease the activity of CBS but does decrease provide a further test of vitamin status. A deficiency the activity of GCT; (2) cysteine sulfinate decarboxy- of pyridoxine does not induce a significant increase in lase, which catalyses taurine formation, also loses its homocysteine under normal conditions [91], though activity rapidly during pyridoxine deficiency; (3) serine it does increase homocysteine, cystathionine and hydroxymethyl transferase (reaction 2, Fig. 1) is another glycine to abnormal levels after a methionine load. This PLP-dependent enzyme. It catalyses the three-carbon suggests that under normal conditions these pyridoxal transfer from serine to tetrahydrofolate to form 5,10- dependent pathways are not limiting [15]. A deficiency methylenetetrahydrofolate (Fig. 1), an important step to of pyridoxine could also inhibit aromatic-l-amino help remethylate homocysteine; (4) PLP is also required acid (dopa) decarboxylase and thereby decrease the for the metabolism of tryptophan to NAD+ as it is a coen- concentration of dopamine in the brain [92]. zyme for kynureninase and kynurenine aminotransferase Pyridoxine appears to be involved in inflammation. that catalyze intermediate reactions [2]; (5) glycine for- In rheumatoid arthritis, vitamin B6 is hypothesized to mation from glyoxylate is also catalyzed by a PLP- be mobilized from the liver and peripheral tissue to the dependent transaminase; (6) vitamin B6 is a coenzyme site of inflammation [90]. There also appears to be an of aminolevulinate synthase and thus B6 deficiency can inverse correlation between PLP and C-reactive protein prevent heme formation and cytochrome c synthesis, in the plasma, independent of homocysteine levels [93], inhibiting the mitochondrial respiration at complex III which has suggested that PLP is used as a coenzyme in of the electron transfer chain (reviewed in this issue [2]). inflammation reactions [94] or that B6 deficiency results The aldehyde group of PLP is much more reactive in inflammation. Smokers and patients with chronic than the aldehyde group of pyridoxal which is mostly inflammation or advanced age have high levels of hydrated. Pyridoxal phosphate binds extensively to pro- interleukin 6 (IL-6) that inhibit pyridoxal phosphatase. teins and enzymes by forming a Schiff base with lysine The decrease in liver PLP would inhibit cystathione ␧-amino groups (Fig. 4) which can inhibit their activity ␤-synthase and moderately increase homocysteine in in vitro e.g. succinic semialdehyde reductase, aldehyde the liver and plasma [95]. reductase, aldose reductase, acetyl-CoA carboxylase Oxalate metabolism is also affected by vitamin B6. activity [88]. Whether this occurs under physiological A vitamin B6 deficient diet induced lipid peroxidation conditions or contributes to pyridoxal induced peripheral in the liver and increased liver lipids, oxalate, calcium sensory neuropathy toxicity is unknown. The depletion and iron levels in rats. This also depleted liver ascor- of perfused liver or hepatocyte pyridoxal phosphate lev- bate, tocopherol and GSH levels and decreased cata- els by ethanol has been attributed to the acetaldehyde lase activity [96]. The increased oxalate levels were metabolite displacing protein lysine bound pyridoxal attributed to decreased PLP-dependent alanine: glyox- phosphate [89]. Most (80%) PLP in the body is in the alate transaminase which increased glyoxalate levels and muscle where it is stored attached to the phosphorylase thus glycolate oxidase activity. Excessive oxalate can- enzyme, and is released during food deprivation, but not not be metabolized and is eliminated by renal excretion. marginal pyridoxine deficiency. PLP binds to albumin High urine oxalate resulted in calcium oxalate accumu- for storage, thus conditions associated with low albumin lation in the kidney, and mitochondrial toxicity [97], loss levels are also likely to show low pyridoxal activity [90]. of the renal function and significant morbidity. Calcium oxalate in the mitochondria can open the mitochon- 4.3. Vitamin B6 deficiency drial proximal tubules, thus leading to swelling of the organelle and inhibition of the mitochondrial electron Vitamin B6 status is commonly measured by deter- transport chain (likely at stage 3) [97]. Pyridoxine ben- mining plasma, erythrocyte or total blood PLP, or efits 50% of patients with primary hyperoxaluria type by determining urinary pyridoxal excretion. It can be I, involving the mistargeting of PLP-dependent alanine: F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 127 glyoxalate transaminase I to mitochondria instead of the pyridoxamine derivatives could act as AGE breakers in peroxisomes [98]. vivo though AGE products are usually thought to remain irreversibly bound to proteins. Collagen cross-linking 4.4. Prevention of oxidative stress and increases with age and is accelerated with diabetes. mitochondrial toxicity by vitamin B6 Pyridoxamine also traps the ␣-dicarbonyl intermediates formed during lipid peroxidation that bind to proteins Vitamin B6 can have a direct antioxidant activity to form ALE without inhibiting fatty acid peroxida- as pyridoxamine was more effective than pyridoxine tion. Pyridoxamine adducts are excreted in the urine at preventing superoxide radical formation, glycated [110,111]. ALE are increased in diabetes and hyper- hemoglobin formation and erythrocyte lipid peroxida- lipidemia and likely contribute to the development of tion during glucose autoxidation [99]. Pyridoxamine was long-term renal and vascular pathology. Currently pyri- also more effective than pyridoxal at preventing H2O2 doxamine is on the FDA “fast track” to phase III clinical induced mitochondrial toxicity in monocytes whereas trials for treatment of diabetic nephropathy [112]. pyridoxal was more effective than pyridoxamine at Ethylene glycol-induced renal mitochondrial toxicity, inhibiting superoxide radical formation [100]. Vitamin as described by inhibition of mitochondrial respiration B6 can also have indirect effects through chelating and loss of mitoxhondrial structure, has been attributed activity. Pyridoxine prevented chromium-induced kid- to its glyoxalate metabolites that can form oxalomalate, ney oxidative stress and toxicity in rat [101]. Pyridoxal an aconitase inhibitor, and oxalic acid, an activator of also induced iron excretion in the rat largely because of the permeability transition pore [97]. The responsive- its iron chelating activity [102] and pyridoxamine pre- ness of renal failure to pyridoxine can be attributed to vented copper induced hepatocyte oxidative stress cyto- increased intracellular PLP binding to the transaminase toxicity [103]. Of particular interest, pyridoxine alone which catalyses glyoxalate conversion to glycine thereby (100 mg/kg oral administration) increased rat kidney preventing the accumulation of glyoxalate and oxalic GSH levels by 57% and ascorbate levels by 64% after acid. Pyridoxine (5 g) is therefore recommended as an 12 h [101]. Supplementary pyridoxine decreases the for- antidote for fatal kidney poisoning by ethylene glycol mation of colon tumors in mice given repeated injections [113]. of azoxymethane [104]. The mechanism is not known, There may be safety risks associated with chronic nor is it known whether the antioxidant properties of excessive pyridoxine administration. Multivitamin sup- B6 result from the reaction of peroxyl radicals with the plements typically provide about 4 mg of B6 per day. hydroxyl and/or the amine groups on the pyridine ring. Symptoms of peripheral sensory neuropathy attributed Vitamin B6 may have antidiabetic antioxidant proper- to B6 toxicity have been observed with chronic dos- ties. Pyridoxine therapy was first described when vitamin ing at or above 50 mg/day for 6–60 months with full supplementation was used to delay peripheral neuropa- recovery after cessation of the treatment [114]. Periph- thy in diabetic patients, presumed to be exposed to eral neuropathy was also induced in dogs following increased oxidative stress [105]. Pyridoxamine also pre- 50–200 mg/kg daily for 40–75 days. vented the development of retinopathy and nephropathy in streptozotocin-induced diabetic rats [106,107]. Indeed 5. Conclusions and future perspectives pyridoxamine is more effective than the antidiabetic agent aminoguanidine at preventing the nonenzymatic The transfer of methyl groups to target biomolecules oxidative glycation of proteins by glucose. This antiox- is a complex process. The molecular carriers are metabo- idant property has been attributed to the amine group of lites of folate and choline, but the formation and activa- pyridoxamine that forms a Schiff base linkage with the tion of these metabolites depends on cobalamin and vita- carbonyls of open-chain sugars, dicarbonyl fragments min B6 as well as other vitamins. As a result, the cellular (e.g. glyoxal and methylglyoxal), Amadori products and pathways for methyl transfers can be disrupted by vita- post-Amadori intermediates [108,109]. Pyridoxamine min deficiencies in more than one way, and detrimental thus not only acts as a scavenger of toxic carbonyl prod- effects resulting from the disruptions can be similar for ucts of glucose and lipid peroxide degradation products the different vitamin deficiencies. There is less overlap which would otherwise covalently bind to proteins, but of pathways for choline than for folate. The details of the also prevents the formation of advanced lipoxidation vitamin B6 and cobalamin pathways and the specificities endproducts (ALE) or glycation endproducts (AGE) by of choline-dependent reactions mean that choline alone blocking oxidative degradation of the Amadori interme- cannot be considered a sufficient supplementation option diate of the Maillard reaction. This activity suggests that for a deficiency of folate, B6, or B12. Furthermore, tar- 128 F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 geted utilization of choline resources for methyl transfer vitamin biomarkers to identify individual vitamin could deflect from other choline-dependent pathways. deficiencies both to provide diagnostic biomark- This review of the scientific literature shows that B ers for examining general pathways and products, vitamins play an essential role in mitochondrial func- and as molecular or genetic tools to understand the tions and suggests that mitochondrial functions are com- consequences and mechanisms of marginal vitamin promised by dietary B vitamin deficiency or increased supply in population studies. For example: Could B vitamin dependency. However the literature is limited studies of polymorphisms provide an aid in the iden- and in many cases based on methods which have since tification of B vitamin requirements? been substantially improved. Further research using (5) Genetic approaches with knock-out murine mod- more refined methods is now needed to define the effects els could be used to better understand specific of vitamin B deficiencies on mitochondrial function over vitamin-dependent reactions and pathways. Knock- a range of environmental and genetic conditions. Some out murine models have been developed for folate, examples of possible approaches are: vitamins B12 and B6 at the level of absorption, metabolism and vitamin-dependent reactions. As (1) New techniques such as proteomics and metabo- folates are substrates for one-carbon transfer reac- nomics could add greatly to our understanding of tions, not for coenzymes, research has thus focused vitamin deficiencies. Vitamin deficiencies can result on absorption and metabolism. Presently there are in the accumulation of potentially reactive inter- knock-out models of Folate binding protein (Folbp1) mediates which can form adducts on circulating and Reduced folate carrier (RFC1) [115] as well as proteins and target tissues. We might ask, for exam- of methylene tetrahydrofolate reductase (Mthfr), a ple: Do B vitamin concentrations affect the levels of central folate metabolic enzyme [116]. Cobalamins sulfolactone adduct concentrations in plasma? Are have also been investigated both for absorption, via there significant differences in the concentration of cubilin receptors [117], and the two cobalamin- these homocysteine adducts in different tissues? Are dependent mammalian enzymes; methionine syn- other adducts formed with other vitamin deficien- thase (MS) [118] and methylmalonyl-CoA mutase cies? (mut) [119]. Vitamin B6 investigations, however, (2) In vitro mammalian cell screening methods could have been limited to an alkaline phosphatase be used to evaluate interactions between B vitamins (TNAP) knock-out [120]. There are a number of fur- and xenobiotics. This might include evaluation of ther genes that could be targeted. Folate is involved protections against cell toxicity induced by xeno- in a very large network of reactions and enzymes biotic toxins, identification of interactions reducing such as serine-hydroxymethyl transferase (transport the levels of B vitamins, and identification of target of the C1 unit across the mitochondrial membrane), subcellular organelles or target different metabolic thymidylate synthase (nucleotide synthesis) or folyl pathways that cause different molecular cytotoxic poly-␥-glutamate synthase (metabolism of dietary mechanisms. For example: Vitamin B6 is reduced folates). Could such genes provide information as by interactions with penicillamine. Are there other to the specificity of folate in the one-carbon path- conditions in which B vitamin activity is lost and is ways in relation to choline? this loss ever tissue specific? (6) The exact role of the two binding proteins, (3) In vitro cellular and in vivo animal studies could transcobalamin and haptocorrin, have in the trans- clarify the degree to which cellular pathways are port of cobalamins in the plasma, membrane recep- compromised by deficiencies and the most effective tors and tissues is not known. This is also the combinations of these factors in reducing possible case for the metabolic activation of vitamin B6 by risk. For example: Is there an optimal combination pyridoxal kinase and pyridoxal phosphate oxidases of folate, vitamin B6 and cobalamin to reduce the would benefit from a genetic approach while PLP- accumulation of homocysteine? What are the opti- dependent enzymes such as ␦-aminolevulinate syn- mal cellular and tissue concentrations of vitamins to thase or transaminases would target specific activi- assure optimal enzyme activity? ties of the vitamin. Are transgenic methods applica- (4) Spontaneous genetic mutations of specific enzymes ble in the study of the metabolism of cobalamin and (polymorphism) could be further investigated to vitamin B6? assess the degree to which daily vitamin require- (7) Some of the current knock-out models are homozy- ments of individuals require a targeted approach. gous lethal, whether at the embryonic [118,121] or There is thus a need to identify further specific post-natal [119,120] stage, which greatly impairs the F. Depeint et al. / Chemico-Biological Interactions 163 (2006) 113–132 129

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