Vitamins B1, B2, B3 and B9 - Occurrence, Biosynthesis Pathways and Functions in Human Nutrition.

BACKGROUND: Vitamins are chemical compounds whose derivatives are involved in vital metabolic pathways of all living organisms. The complete endogenous biosynthesis of vitamins can be performed by many bacteria, yeast and plants, but humans need to acquire most of these essential nutrients with food. In recent years, new types of action of the well-recognized vitamins or their more sophisticated relationships have been reported. CONCLUSION: In this review we present the current knowledge of factors that can influence the yield and regulation of vitamin B1, B2, B3 and B9 biosynthesis in plants which can be important for human nutrition. A summary of modern methods applied for vitamin analysis in biological materials is also provided. Contributions of selected vitamins to the homeostasis of the human organism, as well as their relations to the progress or prevention of some important diseases such as cancer, cardiovascular diseases, diabetes and Alzheimer's disease are discussed in the light of recent investigations. Better understanding of the mechanisms of vitamin uptake by human tissues and possible metabolic or genetic backgrounds of vitamin deficiencies can open new perspectives on the medical strategies and biotechnological processes of food fortification.

Antitumor effect of combined NAMPT and CD73 inhibition in an ovarian cancer model.

Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, ß-methylene adenosine 5'-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors.

Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.

The kynurenine pathway activities in a sub-Saharan HIV/AIDS population.

BACKGROUND: Tryptophan is an essential amino acid for the synthesis of proteins and important metabolites such as serotonin, melatonin, tryptamine and niacin. After protein synthesis, more than 90 % of tryptophan catabolism occurs along the kynurenine pathway. The inflammation-inducible enzyme indoleamine 2,3 dioxygenase (IDO) is responsible for the first rate-limiting step in the kynurenine pathway, i.e., oxidation of tryptophan to kynurenine. Excessive IDO activity in conditions such as HIV/AIDS may lead to tryptophan depletion and accumulation of metabolites downstream from kynurenine. Little is known about the kynurenine pathway of HIV/AIDS patients in sub-Saharan regions. This study, in a low income sub-Saharan HIV/AIDS population, examined the effects of activities in the kynurenine pathway on plasma levels of tryptophan, kynurenine and the neurotoxin quinolinic acid, and on de novo synthesis of nicotinamide. METHODS: Plasma samples were obtained from a cohort of 105 HIV patients and 60 controls. Kynurenine pathway metabolites were analysed using gas chromatography - mass spectrometry. ELISA and flow cytometry were used to assess plasma inflammatory markers. RESULTS: IDO activity, depletion of tryptophan, as well as accumulation of kynurenine and the neurotoxin quinolinic acid, were not only significantly greater in the patients than in the controls, but also markedly greater than in HIV/AIDS patients from developed countries. Tryptophan levels were 12.3 % higher, kynurenine levels 16.2 % lower, quinolinic acid levels 43.2 % lower and nicotinamide levels 27,2 % lower in patients on antiretroviral treatment than in antiretroviral-naïve patients. Patients' kynurenine pathway metabolites correlated with the levels of inflammatory markers, including that of the major IDO-inducer, interferon-gamma. Indications are that the rate of de novo synthesis of nicotinamide in the kynurenine pathway correlates with increases in quinolinic acid levels up to a point where saturation of the enzyme quinolinate phosphoribosyl transferase occurs. CONCLUSIONS: Higher levels of inflammatory activity in this low income sub-Saharan HIV/AIDS population than in patients from developed countries lead to greater tryptophan depletion and greater accumulation of metabolites downstream from tryptophan with quinolinic acid levels often reaching levels associated with the development of HIV/AIDS-associated neurocognitive dysfunction. De novo synthesis of nicotinamide from quinolinic acid contributes to the maintenance of nicotinamide, and by implication NAD levels, in HIV/AIDS patients from low income populations. Antiretroviral treatment partially corrects disturbances in the kynurenine pathway.

A continuous microplate assay for sirtuins and nicotinamide-producing enzymes.

Nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high-throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to alpha-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340 nm. Using this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of k(cat) and K(m) values with the native acetylated substrate acetyl-CoA synthetase-1; measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts; and determination of IC(50) values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide-forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.

Antioxidant metabolism induced by quinic acid. Increased urinary excretion of tryptophan and nicotinamide.

For over 50 years, hippuric/quinic acids were believed to have no biological efficacy. Here data are presented to support the hypothesis that quinic acid is not responsible for any efficacy, but rather that quinic acid nutritionally supports the synthesis of tryptophan and nicotinamide in the gastrointestinal (GI) tract, and that this in turn leads to DNA repair enhancement and NF-kB inhibition via increased nicotinamide and tryptophan production.Moreover, it is shown that quinic acid is a normal constituent of our diet, capable of conversion to tryptophan and nicotinamide via the GI tract microflora, thus providing an in situ physiological source of these essential metabolic ingredients to humans. The concentrations of quinic and hippuric acids in the diet were dependent on each other when analysed in urine, as was evidenced by a significant linear regression analysis that included unsupplemented control subjects (n = 45, p < 0.001). Thus, these ingredients were identified as major dietary components, and not simply originating from environmental pollution as previously had been thought.

Are poly(ADP-ribosyl)ation by PARP-1 and deacetylation by Sir2 linked?

Poly(ADP-ribose) polymerase-1 (PARP-1) safeguards genomic integrity by limiting sister chromatid exchanges. Overstimulation of PARP-1 by extensive DNA damage, however, can result in cell death, as prolonged PARP-1 activation depletes NAD(+), a substrate, and elevates nicotinamide, a product. The decline of NAD(+) and the rise of nicotinamide may downregulate the activity of Sir2, the NAD(+)-dependent deacetylases, because deacetylation by Sir2 is dependent on high concentration of NAD(+) and inhibited by physiologic level of nicotinamide. The Sir2 deacetylase family has been implicated in mediating gene silencing, longevity and genome stability. It is conceivable that poly(ADP-ribosyl)ation by PARP-1, which is induced by DNA damage, could modulate protein deacetylation by Sir2 via the NAD(+)/nicotinamide connection. The possible linkage of the two ancient pathways that mediate broad biological activities may spell profound evolutionary roles for the conserved PARP-1 and Sir2 gene families in multicellular eukaryotes.

Elevation of cerebrospinal fluid choline levels by nicotinamide involves the enzymatic formation of N1-methylnicotinamide in brain tissue.

Nicotinamide administration can elevate plasma and brain choline levels and produce a marginal increase in striatal acetylcholine levels in the rat. We now report that subcutaneous nicotinamide produces a substantial and long-lasting rise in cisternal cerebrospinal fluid (CSF) levels of choline in free-moving rats, possibly through the enzymatic formation of N1-methylnicotinamide (NMN) in brain. CSF choline levels peaked 2 hours after nicotinamide administration and were accompanied by increases in striatal, cortical, hippocampal and plasma choline levels. The enzymatic formation of [3H]NMN in rat brain was evaluated by incubating aliquots of rat brain cytosol with unlabelled nicotinamide and the methyl donor [3H]S-adenosylmethionine. High performance liquid chromatography and radiochemical detection demonstrated that [3H]NMN was specifically formed by a brain cytosolic enzyme. The production of [3H]NMN was dependent on exogenous nicotinamide and could be prevented by denaturing the cytosol. The metabolism of nicotinamide to NMN in rat brain may explain the rise in CSF choline levels since NMN, a quaternary amine, can inhibit choline transport at the choroid villus and reduce choline clearance.

The tellurite-resistance determinants tehAtehB and klaAklaBtelB have different biochemical requirements.

The tehAtehB operon from the Escherichia coli chromosome (32.3 min) mediates resistance to potassium tellurite (K2TeO3) when expressed on a multicopy plasmid such as pUC8 (pTWT100). An MIC of 128 micrograms ml-1 is observed when tehAtehB is expressed in a wild-type host and grown on rich media. In this study, the tehAtehB determinant was transformed into mutants deficient in electron transport processes and/or thiol redox coupling within E. coli. These mutants included ubi, nad, cys, nar, trx, grx, gsh and sod. MICs of tehAtehB transformed into these mutants ranged from 1-16 micrograms K2TeO3 ml-1 compared to 0.03-2 micrograms ml-1 for strains transformed with a control plasmid. The tellurite-resistance determinant locus kilA cloned from the IncP alpha plasmid RK2Ter (pDT1558) was also investigated in these strains. This tellurite-resistance determinant showed little or no dependency on the host genotype. The ability of tehAtehB to mediate resistance in wild-type hosts is limited to rich medium. Rich medium may provide a key unidentified cofactor required by TehATehB that is not provided under minimal conditions. Again, the ability of the kilA determinant to mediate tellurite resistance was independent of medium conditions. These data suggest that either a reducing environment or electron-reducing equivalents are required for tehAtehB to mediate high levels of resistance to potassium tellurite. Therefore, the two resistance determinants studied here possess two very different biochemical mechanisms of resistance. Our data also suggest a mechanism for endogenous resistance to tellurite which involves nitrate reductase, superoxide dismutase, and thiol redox processes.

Nicotinamide methylation and hepatic energy reserve: a study by liver perfusion in vitro.

BACKGROUND/AIMS: The synthesis of pyridine nucleotides from nicotinamide requires adenosine triphosphate. In man when exogenous nicotinamide is poorly utilized in this synthesis, the excess follows a dissipative metabolic pathway and is excreted in urine as N-methylnicotinamide. In human cirrhosis N-methylnicotinamide serum levels are higher than normal, in basal condition and after nicotinamide oral load. The aim of this study was to verify N-methylnicotinamide production in relation to hepatic content of adenosine triphosphate during in vitro perfusion of rat liver, in normal conditions and after adenosine triphosphate depletion by metabolic stress. METHODS: "Stress" was obtained by pre-washing with saline for 15 min before the perfusion with nutritive medium. RESULTS: The adenosine triphosphate decrease in the stressed liver was 38% after pre-washing with saline and 80% at the end of nutritive perfusion. In control liver the corresponding decreases were 1% after pre-washing with nutritive medium and 65% at the end of perfusion with the same medium. The total nicotinamide adenine dinucleotide decreases were 44% and 56% in the stressed liver, and 19% and 52% in the control liver. The output levels of N-methylnicotinamide at 90 min of rat liver nutritive perfusion were 31.50 +/- 4.72 nmol/g for normal liver and 66.40 +/- 13.17 for stressed liver (p<0.001). Liver adenosine triphosphate was inversely related to N-methylnicotinamide production (r=0.93; p<0.001). CONCLUSIONS: These data suggest that nicotinamide methylation may be enhanced when there is hepatic adenosine triphosphate decrease and energy failure induced by hypoxia or metabolic stress, similar to that obtained in vitro by saline washing before perfusion with nutritive medium. This study shows that the evaluation of N-methylnicotinamide production in man (before and after nicotinamide load) might be useful to explore the energy state of diseased liver.

Involvement of NAD-poly(ADP-ribose) metabolism in p53 regulation and its consequences.

We have used two different approaches to study the consequences of NAD/poly(ADP-ribose) deficiency on p53 expression and its activity in V79-derived cell lines. In the first approach, we have used two cell lines that are deficient in poly(ADP-ribose) (pADPR) synthesis because of deficiency in the enzyme poly(ADP-ribose) polymerase (PARP). In a second approach, we have used a cell line that is deficient in NAD/pADPR metabolism due to unavailability of NAD, the substrate for PARP. These NAD/PARP-deficient cell lines exhibit a significant reduction in both baseline p53 expression and its activity compared to their parental V79 cells. Furthermore, etoposide, a topoisomerase II inhibitor that was shown to cause an increase in p53 expression and subsequent apoptosis in V79 cells, failed to produce any significant increase in p53 expression or apoptotic DNA fragmentation in NAD/PARP-deficient cell lines. Thus, our studies suggest that NAD/pADPR synthesis may be involved in the regulation of p53 and its dependent pathways.

Enzyme properties of Aplysia ADP-ribosyl cyclase: comparison with NAD glycohydrolase of CD38 antigen.

An ecto-enzyme of NAD glycohydrolase (NADase) induced by retinoic acid in HL-60 cells is attributed to the molecule of CD38 antigen [Kontani, K., Nishina, H., Ohoka, Y., Takahashi, K., and Katada, T. (1993) J. Biol. Chem. 268, 16895-16898]. CD38 antigen has an amino acid sequence homologous to Aplysia ADP-ribosyl cyclase which generates cyclic adenosine diphosphoribose (cADPR) and nicotinamide (NA) from beta-NAD+. On the basis of this sequence homology, we compared enzyme properties between CD38 NADase expressed as a fusion protein in Escherichia coli and ADP-ribosyl cyclase purified from the ovotestis of Aplysia kurodai. 1) beta-NAD+ analogs, nicotinamide 1, N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide, did not serve as good substrates for the ADP-ribosyl cyclase, suggesting that the intact adenine ring of beta-NAD+ was required for the cyclase-catalyzed reaction. On the other hand, CD38 NADase utilized the NAD analogs to form ADP-ribose and NA. 2) Kinetic analyses of the ADP-ribosyl cyclase reaction revealed that NA was first released from the substrate (beta-NAD+)-enzyme complex, followed by the release of another product, cADPR, which was capable of interacting with the free enzyme. 3) The enzyme reaction catalyzed by the ADP-ribosyl cyclase was fully reversible; beta-NAD+ could be formed from cADPR and NA with a velocity similar to that observed in the degradation of beta-NAD+. However, CD38 NADase did not catalyze the reverse reaction to form beta-NAD+ from ADP-ribopase and NA. 4) The CD38 NADase activity was, but the ADP-ribosyl cyclase activity was not, inhibited by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-activity relationships for tumour radiosensitization by analogues of nicotinamide and benzamide.

Nicotinamide has been shown in our laboratory and those of other investigators to be an effective radiosensitizer of a variety of mouse tumours, while producing little or no radiosensitization of normal tissues. Its mechanism of action is different from classical electron-affinic compounds and appears to be the result of improved tumour oxygenation. In this study we have synthesized 29 analogues of nicotinamide and benzamide and characterized them for their tumour radiosensitization and acute toxicity in mice. The data show that a wide range of additions to the nicotinamide and benzamide ring produce tumour radiosensitization similar to that produced by equimolar doses of misonidazole, but that substitutions of the amide tend to reduce radiosensitization. Other structure-activity relationships are evident. Although some compounds produce similar tumour radiosensitization to nicotinamide at equimolar doses, and are comparably low in acute toxicity, none appears sufficiently superior to supplant nicotinamide itself as a candidate for clinical trials. Thus these data provide evidence that nicotinamide, because of the extensive experience with its use in man, is likely to be the best drug in the benzamide-nicotinamide series for development as a radiosensitizer of human tumours.

Mechanism of action of choleragen. Evidence for ADP-ribosyltransferase activity with arginine as an acceptor.

Choleragen catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide; nicotinamide production was dramatically increased by L-arginine methyl ester and to a lesser extent by D- or L-arginine, but not by other basic amino acids. Guanidine was also effective. Nicotinamide formation in the presence of L-arginine methyl ester was greatest under conditions previously shown to accelerate the hydrolysis of NAD by choleragen (Moss, J., Manganiello, V. C., and Vaughan, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4424-4427). After incubation of [adenine-U14C]NAD and L[3H]arginine with coleragen, a product was isolated by thin layer chromatography that contained adenine and arginine in a 1:1 ratio and has been tentatively identified as ADP-ribose-L-arginine. Parallel experiments with [carbonyl-14C]NAD have demonstrated that formation of the ADP-ribosyl-L-arginine derivative was associated with the production of [carbonyl-14C]nicotinamide. As guanidine itself was active and D- and L-arginine was equally effective in promoting nicotinamide production, whereas citrulline, which possesses a ureido rather than a guanidino function, was inactive, it seems probable that the guanidino group rather than the alpha-amino moiety participated in the linkage to ADP-ribose. Based on the assumption that the ADP-ribosylation of L-arginine by choleragen is a model for the NAD-dependent activation of adenylate cyclase by choleragen, it is proposed that the active A protomer of choleragen catalyzes the ADP-ribosylation of an arginine, or related amino acid residue in a protein, which is the cyclase itself or is critical to its activation by choleragen.

Isolation of a metabolite capable of differentially supporting the growth of nicotinamide adenine dinucleotide auxotrophs of Escherichia coli.

A compound, isolated from the culture fluid of a nadC auxotroph of Escherichia coli grown in a minimal medium, supports the growth of both a nadA and a nadB mutant. This metabolite exhibits an ultraviolet light absorption spectrum and a mass spectrum, different from quinolinic acid. This compound may be the precursor of quinolinic acid, an intermediate in the biosynthesis of nicotinamide adenosine dinucleotide.