Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.     

The Regulatory Role of NAD in Human and Animal Cells

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes in the redox reactions of various essential metabolic pathways. NAD⁺ also serves as a substrate for several families of regulatory proteins, such as protein deacetylases (sirtuins), ADP-ribosyltransferases, and poly(ADP-ribose) polymerases, that control vital cell processes including gene expression, DNA repair, apoptosis, mitochondrial biogenesis, unfolded protein response, and many others. NAD⁺ is also a precursor for calcium-mobilizing secondary messengers. Proper regulation of these NAD-dependent metabolic and signaling pathways depends on how efficiently cells can maintain their NAD levels. Generally, mammalian cells regulate their NAD supply through biosynthesis from the precursors delivered with the diet: nicotinamide and nicotinic acid (vitamin B3), as well as nicotinamide riboside and nicotinic acid riboside. Administration of NAD precursors has been demonstrated to restore NAD levels in tissues (i.e., to produce beneficial therapeutic effects) in preclinical models of various diseases, such as neurodegenerative disorders, obesity, diabetes, and metabolic syndrome.     

Decrease in ADP-ribosylation of HeLa non-histone proteins from interphase to metaphase

Variations for non-histones in the ADP-ribosylating activities of interphase and metaphase cells were investigated. 32P-Labeled nicotinamide adenine dinucleotide ([32P]NAD), the specific precursor for the modification, was used to radioactively label proteins. Permeabilized interphase and mitotic cells, as well as isolated nuclei and chromosomes, were incubated with the label. One-dimensional and two-dimensional gels of the proteins of total nuclei and chromatin labeled with [32P]NAD showed more than 100 modified species. Changing the labeling conditions resulted in generally similar patterns of modified proteins, though the overall levels of incorporation and the distributions of label among species were significantly affected. A less complex pattern was found for nuclear scaffolds. The major ADP-ribosylated proteins included the lamins and poly(ADP-ribose) polymerase. Inhibitors of ADP-ribosylation were effective in preventing the incorporation of label by most non-histones. Snake venom phosphodiesterase readily removed protein-bound 32P radioactivity. A fundamentally different distribution of label from that of interphase nuclei and chromatin was found for metaphase chromosome non-histones. Instead of 100 or more species, the only major acceptor of label was poly(ADP-ribose) polymerase. This profound change during mitosis may indicate a structural role for ADP-ribosylation of non-histone proteins.     

Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans

NAD+ is essential for life in all organisms, both as a coenzyme for oxidoreductases and as a source of ADPribosyl groups used in various reactions, including those that retard aging in experimental systems. Nicotinic acid and nicotinamide were defined as the vitamin precursors of NAD+ in Elvehjem's classic discoveries of the 1930s. The accepted view of eukaryotic NAD+ biosynthesis, that all anabolism flows through nicotinic acid mononucleotide, was challenged experimentally and revealed that nicotinamide riboside is an unanticipated NAD+ precursor in yeast. Nicotinamide riboside kinases from yeast and humans essential for this pathway were identified and found to be highly specific for phosphorylation of nicotinamide riboside and the cancer drug tiazofurin. Nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide riboside is a useful compound for elevation of NAD+ levels in humans.     

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.     

Pyridine nucleotide synthesis in 3T3 cells.

The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12-fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5-fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.     

Dependence of ADP-ribosylation of histones of chicken liver nuclei on the intranuclear content of NAD

The dependence of ADP-ribosylation of chicken liver nuclear histones on NAD concentration in the nuclei was studied under conditions of stimulation of coenzyme synthesis by the nicotinamide and nicotinic acid as well as upon addition of various concentrations of the [Ado-U-14C]NAD nuclei to the incubation mixture. In the first case, the rate of [Ado-U-14C]NAD incorporation into the histones was decreased due to the dilution of the label by the de novo synthesized NAD. The amount of the latter formed under effects of nicotinic acid and nicotinamide increased, correspondingly, from 2,2 X 10(-5) mmol up to 4.1 X 10(-5) and 7.0 X 10(-5) mmol per mg of nuclear protein. The incorporation of [Ado-U-14C]NAD into the histones decreased from 12.0 X 10(-8) mmol after incubation of liver slides with nicotinic acid and nicotinamide down to 8.0 X 10(-8) and 7.0 X 10(-8) mmol, respectively. With a rise in the concentration of exogenous [Ado-U-14C]NAD, the level of ADP-ribosylation of nuclear histones increased, the plot [14C]NAD incorporation at the labeled coenzyme concentration of 25 X 10(-7) mM/mg of histone had a plateau. Changes in the labeled substrate concentration brought about corresponding changes in the average length of the histone-linked poly-(ADP-ribose) chain.     

Murine Glial Cells Regenerate NAD, After Peroxide-Induced Depletion, Using Either Nicotinic Acid, Nicotinamide, or Quinolinic Acid as Substrates

Abstract: The potential for regeneration of intracellular pyridine nucleotide levels from different precursors, after peroxide-induced NAD depletion, in cultured glial cells was investigated. Cultured murine glial cells showed a decrease in intracellular NAD levels of >40% after treatment with H₂O₂ (100 µ M ). Removal of the H₂O₂ followed by a 2-h incubation did not result in NAD recovery in the absence of precursors. However, NAD levels increased significantly in these cells after the following substrate additions, at minimum effective concentrations of 1 m M for quinolinic acid (QUIN), 500 µ M for nicotinamide, and 2 µ M for nicotinic acid. The regeneration of significant amounts of NAD from nicotinic acid at doses 250 and 500 times lower than either nicotinamide or QUIN indicates a preferred route for NAD biosynthesis in glial cells in vitro, probably via nicotinic acid phosphoribosylation.     

Formation of the N'-methylnicotinamide adenine dinucleotide derivative of NAD in intact rat pituitary tumor GH3 and human promyelocytic leukemia HL-60 cells

The NAD analog N'-methylnicotinamide adenine dinucleotide (N'AD) is formed in intact human promyelocytic leukemia HL-60 and in rat pituitary tumor GH3 cells during treatment of the cultured cells with the nicotinamide derivative N'-methylnicotinamide (N'CH3NAm). N'AD formation is associated with the induced maturation of HL-60 cells and increased hormone production by GH3 cells during treatment with the nicotinamide derivative. N'AD is detected by HPLC analysis of cytoplasmic extracts as a peak which elutes near NAD. Four facts indicate that this compound is N'AD. First, a compound which elutes with identical time retention is produced by transglycosylation during reaction of NAD with pig brain NAD glycohydrolase in the presence of excess N'CH3NAm. Second, the putative N'AD is degraded by prolonged digestion with the NAD glycohydrolase to ADP-ribose. Third, N'AD formation is prevented by addition of nicotinamide along with N'CH3NAm to compete with binding of N'CH3NAm to the NAD glycohydrolase. Fourth, radioactive precursor labeling demonstrates that it contains adenosine, but it is not labeled by radioactive nicotinamide. The biological relevance of N'AD formation was evaluated. The appearance of N'AD precedes development of HL-60 maturation, and NAD levels increase, not decrease, as observed in other cell types, during treatment with N'CH3NAm. Therefore, we propose that N'AD, not the pyridine base itself, is the active species in inducing maturation. The results provide support of a role for NAD metabolism, probably ADP-ribosylation, in the regulation of HL-60 maturation and in hormone production by pituitary cells.     

Importance of nicotinamide as an NAD precursor in the human erythrocyte

The effect of variation in the concentration of inorganic phosphate and of the pyridine precursors nicotinamide (NAm) and nicotinic acid (NA) on pyridine nucleotide synthesis was studied using intact human erythrocytes. A wide range of incubation times was employed. The results showed that under physiological conditions the rate of synthesis of NAD from NAm exceeded that from NA twofold, while the reverse situation pertained at higher and unphysiological substrate levels. The two pathways had different regulation points. For NAm the rate-limiting factor was the initial step, namely its conversion into the mononucleotide, while for NA it lay at the second step, conversion of NA mononucleotide (NAMN) to its adenine dinucleotide. At physiological substrate levels the uptake of NA and conversion to NAMN were rapid, while the uptake and conversion of NAm were time dependent. This process was stimulated significantly by inorganic phosphate only for NAm. These results indicate that while NA is the predominant precursor of human erythrocyte NAD at high (unphysiological) substrate and phosphate levels, NAm is more efficient as an NAD precursor under physiological conditions, suggesting an important and hitherto unrecognized role for nicotinamide in NAD synthesis in vivo.     

NAD+ levels control Ca2+ store replenishment and mitogen-induced increase of cytosolic Ca2+ by Cyclic ADP-ribose-dependent TRPM2 channel gating in human T lymphocytes

Intracellular NAD(+) levels ([NAD(+)](i)) are important in regulating human T lymphocyte survival, cytokine secretion, and the capacity to respond to antigenic stimuli. NAD(+)-derived Ca(2+)-mobilizing second messengers, produced by CD38, play a pivotal role in T cell activation. Here we demonstrate that [NAD(+)](i) modifications in T lymphocytes affect intracellular Ca(2+) homeostasis both in terms of mitogen-induced [Ca(2+)](i) increase and of endoplasmic reticulum Ca(2+) store replenishment. Lowering [NAD(+)](i) by FK866-mediated nicotinamide phosphoribosyltransferase inhibition decreased the mitogen-induced [Ca(2+)](i) rise in Jurkat cells and in activated T lymphocytes. Accordingly, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores was greatly reduced in these cells in the presence of FK866. When NAD(+) levels were increased by supplementing peripheral blood lymphocytes with the NAD(+) precursors nicotinamide, nicotinic acid, or nicotinamide mononucleotide, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores as well as cell responsiveness to mitogens in terms of [Ca(2+)](i) elevation were up-regulated. The use of specific siRNA showed that the changes of Ca(2+) homeostasis induced by NAD(+) precursors are mediated by CD38 and the consequent ADPR-mediated TRPM2 gating. Finally, the presence of NAD(+) precursors up-regulated important T cell functions, such as proliferation and IL-2 release in response to mitogens.     

Nicotinamide adenine dinucleotide metabolism in Candida albicans.

The functional pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis and their regulation were studied in the dimorphic fungus Candida albicans. The presence of a functional endogenous pathway of NAD biosynthesis from tryptophan was demonstrated. In addition, nicotinamide served as an efficient salvage precursor for NAD biosynthesis but nicotinate was not utilized. The pathway for nicotinamide utilization involved nicotinate and nicotinate nucleotides as intermediates, suggesting that the failure to utilize nicotinate involves a transport defect. The mechanisms that regulate NAD levels during exponential growth operated to maintain constant NAD levels when NAD biosynthesis occurred exclusively from endogenous or salvage pathways or from a combination of the two. The regulation also operated such that the salvage pathway was preferentially utilized.     

Reactivation of NAD(H) biosynthetic pathway by exogenous NAD+ in Nil cells severely depleted of NAD(H)

The culture of Nil hamster fibroblasts in MEM lacking nicotinamide (NAm-MEM) leads to: (1) the rapid loss of intracellular total nicotinamide adenine dinucleotide (NAD(H)) content in these cells from a level of 150-200 pmoles/10(5) cells to less than 20 pmoles/10(5) cells; (2) the cessation of cell division and inhibition of DNA synthesis; and (3) a reduction of glucose consumption and lactic acid production. In most situations, following nicotinamide starvation, the restoration of intracellular NAD(H) follows rapidly the readdition of NAD+ (oxidized), nicotinamide mononucleotide (NMN), nicotinamide, or nicotinic acid. Resumption of cell division occurs after only a lag of about 24 hours. Nil cells subcultured for three consecutive times in the absence of nicotinamide (3(0) NAm- cells) exhibit different behavior. These severely starved cells are incapable of quickly restoring their intracellular NAD(H) content to normal levels when provided with any pyridine ring compound except NAD+. One-hour exposure of such cells to NAD+ allows utilization of nicotinamide to rapidly restore intracellular NAD(H). This short incubation with NAD+ does not result in any significant restoration of intracellular NAD(H) or lead to the accumulation of an intracellular pool of some precursor. This function of NAD+ as a stimulatory signal to the NAD(H)-biosynthetic pathway in severely starved Nil cells is a previously unreported role of NAD+, and does not require protein synthesis.     

Turnover at nicotinamide adenine dinucleotide in cultures of human cells.

The rate of turnover of nicotinamide adenine dinucleotide (NAD) in the human cell line, D98/AH2, has been estimated by measuring the rates of entry into and exit from NAD molecules of 14C-adenine. In one set of experiments, cells were labeled by growth in medium containing 14C-adenine for six hours and then shifted to medium without labeled adenine. The loss of 14C-adenine from the adenine nucleotide and pyridine nucleotide pools was measured, and the data were analyzed using an analytical treatment which corrects for the relatively slow turnover of precursor pools. The loss of 14C-adenine from the NAD pool and from the precursor ATP pool could be related to the absolute rate of NAD breakdown. Under the experimental conditions used, the rate of NAD turnover ranged from 83,000 to 126,000 molecules per second per cell. In a complementary experiment cells were grown in the presence of unlabeled adenine, then shifted into medium containing 14C-adenine and the rate of entry of 14C-adenine into adenine and pyridine nucleotides was measured. The data were treated using a similar analysis to relate the rate of entry of 14C-adenine into NAD and the precursor ATP pools to the absolute turnover rate of NAD. This analysis gave a value for NAD turnover of 78,000 molecules per second per cell in excellent agreement with results from the pulse-chase experiments. The results from both types of experiment indicate that within D98/AH2 cells the half-life of an intact NAD molecule is 60 +/- 18 minutes. Thus, in a human D98/AH2 cell growing with a generation time of 24 hours, NAD is turning over at twice the rate found in Escherichia coli with a generation time of half an hour.     

Metabolism of NAD and N1-methylnicotinamide in growing and growth-arrested cells

Nicotinamide is metabolized primarily into NAD and N1-methylnicotinamide in cultured cells of normal rat kidney. The metabolic pathways for the nicotinamide metabolites are independently regulated and are influenced by the growth stage of the cells. N1-Methylnicotinamide levels are 1.5--2-fold elevated in cells growth-arrested by treatment with histidinol, thymidine, or picolinic acid, or by serum starvation. This increase is due to a more rapid rate of synthesis rather than decrease in excretion. The rates of both synthesis and degradation of NAD are increased in serum-starved cells so that the NAD concentration is the same as it is in growing cells. NAD and N1- methylnicotinamide levels are not significantly increased when the intracellular nicotinamide concentration is increased 20-fold by addition of excess nicotinamide to the culture medium, demonstrating that the size of the nicotinamide pool does not limit synthesis of these compounds. In medium containing normal amounts of nicotinamide, the apparent first-order rate constant for the decay of NAD, radioactively labeled in the nicotinamide moiety, is about 4 h-1. Labeled N1-methylnicotinamide is not metabolized, but rather is excreted into the medium with a first-order rate constant of 3.9 h-1. The rate of loss of label from NAD, but not from N1-methylnicotinamide, is increased about twofold by addition of excess nicotinamide to the culture medium. This could be explained by a dilution of a labeled nicotinamide pool which is formed during NAD degradation and which is recycled into NAD but not into N1-methylnicotinamide. The results demonstrate a rapid turnover of NAD at the bond joining nicotinamide and ADP-ribose, in agreement with previous studies. In addition, the results show that nicotinamide is metabolized into N1-methylnicotinamide with what appears to be a carefully regulated synthetic mechanism. The existence of significant amounts of N1-methylnicotinamide in cultured cells raises the question of the physiological importance of this compound.     

Synthesis of stable isotope-labeled precursors for the biosyntheses of capsaicinoids, capsinoids, and capsiconinoids

Stable isotope-labeled precursors were synthesized for an analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the biosynthetic flow of capsaicinoids, capsinoids, and capsiconinoids. [1'-(13)C][5-(2)H]-Vanillin was prepared by the condensation of guaiacol with [(13)C]-chloroform and a D(2)O treatment. Labeled vanillylamine, vanillyl alcohol, ferulic acid, and coniferyl alcohol were prepared from the labeled vanillin. The labeled vanillylamine was converted to labeled capsaicinoid in a crude enzyme solution extracted from pungent Capsicum fruits.     

The control of nucleic acid and nicotinamide nucleotide synthesis in regenerating rat liver

1. The effect of injecting nicotinamide on the incorporation of [(14)C]orotate into the hepatic nucleic acids of rats after partial hepatectomy was investigated. 2. At 3h after partial hepatectomy the rapid incorporation of [(14)C]orotate into RNA, and at 20h after partial hepatectomy the incorporation of [(14)C]orotate into both RNA and DNA, were inhibited in a dose-dependent fashion by the previous injection of nicotinamide. 3. The injection of nicotinamide at various times before the injection of [(14)C]orotate at 20h after partial hepatectomy revealed an inhibition of the incorporation of orotate into RNA and DNA which was non-linear with respect to the duration of nicotinamide pretreatment. 4. The induction of a hepatic ATP depletion by ethionine demonstrated that the synthesis of hepatic NAD and NADP in partially hepatectomized rats was more susceptible to an ATP deficiency than in control rats. 5. The total hepatic activity of ribose phosphate pyrophosphokinase (EC 2.7.6.1) was assayed at various times after partial hepatectomy and found to be only marginally greater than the maximum rate of hepatic NAD synthesis induced in vivo by nicotinamide injection between 12 and 24h after partial hepatectomy. 6. It is suggested that a competition exists between NAD synthesis and purine and pyrimidine nucleotide synthesis for available ATP and particularly 5-phosphoribosyl 1-pyrophosphate. In regenerating liver the competition is normally in favour of the synthesis of nucleic acid precursors, at the expense of NAD synthesis. This situation may be reversed by the injection of nicotinamide with a subsequent inhibition of nucleic acid synthesis.     

Cytoprotective effects of nicotinamide derivatives in endothelial cells

Following discovery of NAD(+)-dependent reactions that control gene expression, cytoprotection, and longevity, there has been a renewed therapeutic interest in precursors, such as nicotinamide and its derivatives. We tested 20 analogues of nicotinamide for their ability to protect endothelial cells from peroxynitrite stress and their effect on poly (ADP-ribose) polymerase (PARP) activity. Several nicotinamide derivatives protected endothelial cells from peroxynitrite-induced depletion of cellular NAD(+) and ATP concentrations, but only some of these compounds inhibited PARP. We conclude that some nicotinamide derivatives provide protection of endothelial cells against peroxynitrite-induced injury independent of inhibition of PARP activity. Preservation of the NAD(+) pool was a common effect of these compounds.     

Nicotinamide prolongs survival of primary cultured hepatocytes without involving loss of hepatocyte-specific functions

When hepatocytes isolated from adult rats were cultured in the presence of 10 mM nicotinamide, insulin- and epidermal growth factor-induced DNA synthesis and cell proliferation were found to be greatly stimulated, and the cells were able to be kept alive for more than one month. In the nicotinamide-treated hepatocytes, albumin and tryptophan 2,3-dioxygenase mRNAs were present at much higher levels than in the untreated control, and the inducibility of tryptophan oxygenase gene expression by dexamethasone and glucagon was also preserved. Without nicotinamide, primary cultured hepatocytes were viable for only 5-7 days and the hepatocyte-specific phenotypes were rapidly lost. The intracellular NAD level was maintained in the nicotinamide-treated hepatocytes at or above the level in intact liver but depleted in hepatocytes without nicotinamide. These results suggest that the maintenance of the intracellular NAD level is essential for the growth and functioning of hepatocytes and that nicotinamide can preserve the NAD level by blocking NAD degradation as well as by acting as a precursor for NAD synthesis.     

Stable isotope dilution-based accurate comparative quantification of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo (15)N-isotope enrichment

Stable isotope dilution-based comparative quantification of nitrogen-containing metabolites for highly sensitive and selective metabolomics was developed using liquid chromatography/mass spectrometry (LC/MS) and (15)N-isotope enrichment. We produced metabolically stable isotope-labeled Arabidopsis T87 cells by culturing with (15)N-labeled medium. We found that the growth of cells maintained in (15)N-labeled medium is very similar to the growth in normal medium, as evidenced by cell morphology, doubling time, and measurement of chlorophyll and carotenoid contents. Complete incorporation of (15)N in folate, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) in T87 cells was accomplished after culturing for 21 d. Accurate comparative quantification of folate, SAM, and SAH was established by means of LC/MS using the isotopomers of the target metabolites as internal standards. The within- and between-run assay coefficients of variation for the folate, SAM, and SAH levels were all less than 8.5%. Stable isotope labeling by nitrogen source in Arabidopsis T87 cell culture provided simple, inexpensive, and accurate amino acid profiling. This interesting new protocol is valuable for the study of dynamic changes in N-compound pools in cultured cells.