The incorporation of [5,6-13C2]Nicotinic acid into the tobacco alkaloids examined by the use of 13C nuclear magnetic resonance

[5,6-¹⁴C,¹³C₂]Nicotinic acid was prepared from [¹⁴C,¹³C]methyl iodide via nitromethane, 2-nitroacetaldehyde oxime, 3-nitroquinoline, 3-aminoquinoline, and quinoline in 20% overall yield. Administration of this material to Nicotiana tabacum and N. glauca afforded labeled anabasine, anatabine, nicotine, and nornicotine. Qualitative and quantitative incorporation (0.07–4.5% specific incorporation) was determined by radioactive assay and by examination of the ¹³C NMR spectra of these alkaloids. Satellites due to spin-spin coupling of the incorporated contiguous ¹³C atoms were observed at the resonances due to C-5 and C-6 in anabasine, nicotine, and nornicotine. In anatabine, satellites were found at C-5, C-6, C-5′, and C-6′.     

The incorporation of ornithine-[2,3-13C2] into nicotine and nornicotine established by NMR

dl-Ornithine-[2,3-¹³C₂] was synthesized from acetate-[1-¹³C] and ethyl acetamidocyanoacetate-[2-¹³C]. This labelled material was mixed with dl-ornithine-[5-¹⁴C] and fed to Nicotiana glutinosa plants by the wick method. After 10 days the plants were harvested affording radioactive nicotine and nornicotine (0.14% and 0.051% specific incorporations, respectively). Even at these low specific incorporations an examination of their ¹³C NMR spectra established the incorporation of ornithine symmetrically into the pyrrolidine rings of these alkaloids. Satellites were observable at the signals due to C-2′, 3′, 4′ and 5′ positions, arising by the presence of contiguous carbons at C-2′, 3′ and C-4′, 5′.     

Metabolism of nicotine in Nicotiana glauca

A mixture of (?)-nicotine-[2′-³H] and (±)-nicotine-[2′-¹⁴C] was administered to Nicotiana glauca plants for 3 days, resulting in the formation of radioactive nornicotine (49·5% incorporation) and myosmine (2·05% incorporation). Negligible activity was detected in anabasine, cotinine, or 3-acetylpyridine, the last two compounds being added as carriers to the harvested plants. The radioactive nornicotine consisted of 48% (?)-nornicotine-[2′-¹⁴C,³H] and 52% (+)-nornicotine-[2′-¹⁴C]. Thus if (+)-nornicotine is formed from (?)-nicotine the transformation must involve loss of the hydrogen from C-2′. Myosmine is presumably formed from nicotine via nornicotine. However by feeding myosmine-[2′-¹⁴C] to N. glauca it was shown that the dehydrogenation is not reversible, no activity being detected in nornicotine. Nicotinic acid (0·14% incorporation) was a metabolite of myosmine-[2′-¹⁴C]. Essentially all the activity of the nicotinic acid was located on its carboxyl group, indicating that myosmine was a direct precursor.     

Spermidine: an indirect precursor of the pyrrolidine rings of nicotine and nornicotine in Nicotiana glutinosa

Spermidine, which was labeled asymmetrically in its four-carbon moiety ([6-¹⁴C]-1,5,10-triazadecane), was administered to Nicotiana glutinosa plants. After 7 days the plants were harvested, yielding radioactive nicotine (0.43 % incorporation) and nornicotine (0.07 % inc.). A systematic degradation of the alkaloids indicated that they were labelled equally at C-2′ and C-5′ of their pyrrolidine rings. These results are consistent with the hypothesis that spermidine is degraded to putrescine prior to its incorporation into the pyrrolidine rings of nicotine and nornicotine.     

Lipogenesis in Fatty Liver by Amino Acid Imbalance

Lipogenesis in the fatty liver of rat which was induced by feeding an amino acid-irnbalanced diet containing 8% casein supplemented with 0.3% dl-methionine has been investigated by measuring the incorporation of acetate-1-¹⁴C into various lipid fractions during in vitro incubation of liver slices.

In the incorporation of acetate-1-¹⁴C into the total lipid per one g of the slices, no significant difference for the imbalance group was observed. However, the total radioactivity of liver lipid per 100 g of the body and the incorporation into triglyceride in the lipids were significantly higher in the imbalance group than in the control group. Conversion of acetate-1-¹⁴C to CO₂ was not impaired in the imbalance group.

It is evident from these results that the induction of this type of fatty liver is due mainly to the synthesis of triglyceride.     

Nature of DNA and RNA Degradation in Escherichia coli Induced by Colicin E2

Lipogenesis in the fatty liver of rat, which was induced by feeding an amino acid unbalanced diet containing 8% casein supplemented with 0.3% dl-methionine, has been studied by measuring the incorporation of glycerol-1-¹⁴C, palmitate-1-¹⁴C, citrate-1,5-¹⁴C, pyruvate-1-¹⁴C and pyruvate-2-¹⁴C into various lipid fractions and ¹⁴CO₂ during in vitro incubation of liver slices.

The total radioactivity of liver lipid per 100 g of the body and the incorporation of each substrate into triglyceride in the lipid were significantly higher in the imbalance group than the control group. Conversion of each substrate to ¹⁴CO₂ was not imparied in the imbalance group.

It is evident from these results that the induction of this type of fatty liver is due mainly to the triglyceride synthesis by both the fatty acid synthesis and the transesterification of fatty acid.

These results are considered to support the previous assumption in which acetate-1-¹⁴C was used as a precursor.     

Fast track to the trichome: induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda

 Nicotiana repanda Wildenow ex Lehmann acylates nornicotine in its trichomes to produce N-acyl-nornicotine (NacNN) alkaloids which are dramatically more toxic than nicotine is to the nicotine-adapted herbivore, Manduca sexta. These NacNNs, like nicotine, were induced by methyl jasmonate (MeJA) and wounding, but the 2-fold increase in NacNN pools was much faster (within 6 h) than the MeJA-induced increase in nornicotine pools (24 h to 4 d), its parent substrate. When ¹⁵NO₃ ⁻ pulse-chase experiments with intact and induced plants were used to follow the incorporation of ¹⁵N into alkaloids in different plant parts over the plant's lifetime, it was found that the root nicotine pool was most rapidly labeled, followed by the shoot nornicotine and NacNN pools. After 3 d, 3.12% of ¹⁵N acquired was in nicotine (0.93%), nornicotine (0.32%) and NacNNs (1.73%) while only 0.14% was in anabasine. Once NacNNs are externalized to the leaf surface, they are not readily re-distributed within the plant and are lost with senescing leaves. The wound- and MeJA-induced N-acylation of nornicotine is independent of induced changes in nornicotine pools and the rapidity of the response suggests its importance in defense against herbivores. Received: 3 July 1999 / Accepted: 17 September 1999     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Biosynthesis of securinine,the main alkaloid of Securinega suffruticosa

Biosynthesis of securinine was studied by incorporation experiments in Securinega suffruticosa. Among presumed precursors tested, lysine, cadaverine, and tyrosine showed the highest incorporation into securinine. Degradation experiments revealed that cadaverine-[1,5-¹⁴C] labelled specifically the piperidine ring of securinine and the radioactivity from dl-tyrosine-[2-¹⁴C] was introduced into the C-11 lactone carbonyl. Experiments with L-tyrosine-[U-¹⁴C] and L-tyrosine-[3′,5′-³H; U-¹⁴C] prove that the remaining C₆Sz.sbnd;C₂ moiety is derived from the aromatic ring and the C-2 and C-3 or tyrosine.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

Evidence for arginine as the endogenous precursor of necines in heliotropium

In pyrrolizidine alkaloid-bearing Heliotropium angiospermum and H. indicum shoots exposed, in the light, to ¹⁴C-labeled CO₂ for 44 hours, the incorporation of ¹⁴C into 1,2-epoxy-1-hydroxymethylpyrrolizidine and retronecine amounted to 0.23 and 0.15%, respectively, of the total carbon assimilated. Treatment of the shoots with α-dl-difluoromethylornithine, the specific ornithine decarboxylase inhibitor, at 1 to 2 millimolar had no effect on ¹⁴C incorporation into the necines. In contrast, α-dl-difluoromethylarginine, the specific arginine decarboxylase inhibitor, prevented the incorporation of ¹⁴C into the necines of both species; the inhibitor did not affect the absolute incorporation of ¹⁴C from exogenous [1,4-¹⁴C] putrescine in either species. Thus, arginine is the only apparent endogenous precursor of the putrescine channeled into pyrrolizidines, at least in these two Heliotropium species that exhibited a relatively much higher in vitro activity of arginine decarboxylase than of ornithine decarboxylase. However, within 28 hours after administration, not only exogenous l-[5-¹⁴C]arginine, but also exogenous l-[5-¹⁴C]ornithine exhibited significant incorporation of their label into the necines, incorporation that could be partially prevented by both inhibitors. Neither inhibitor affected the rates of ¹⁴C-labeled CO₂ assimilation, transformation of labeled assimilates into ethanol-insoluble compounds, or the very high degree of conversion of the introduced amino acids into other compounds. Methodology related to alkaloid biosynthetic studies is discussed.     

The enzymic preparation of 14C-kaurene

Summary Endosperm from immature seeds of Cucurbita pepo L. converts 2-¹⁴C-dl-mevalonate to ¹⁴C-(-)-kaurene with a yield of nearly 40% of the active isomer. Kaurene is the main product and the only diterpene hydrocarbon which is formed from mevalonate in the system and is therefore easily obtained radiochemically pure. The product was identified by thin-layer chromatography and recrystallization with authentic (-)-kaurene to constant specific radioactivity.     

Selective Inhibition of the Demethylation at C-14 in Ergosterol Biosynthesis by the Fungicide,Denmert (S-1358)

In cell-free homogenates of Saccharomyces cerevisiae, Denmert (S-1358) inhibited the incorporation of radioactivity from dl-mevalonate-2-¹⁴C into 14-desmethyl-lanosterol, 4α-methyl-cholesta-8,24-dien-3-one, 4α-methyl-zymosterol and 4-desmethyl sterols (zymosterol and episterol) at a concentration of 10^?4 m. Concomitantly, a large accumulation of radioactivity was observed in the Ianosterol fraction.

In good agreement with the results described above, Denmert inhibited the conversion of ¹⁴C-labeled lanosterol to 4-desmethyl sterols, while the conversion of ¹⁴C-labeled 14-desmethyl-lanosterol to 4-desmethyl sterols was hardly affected by the fungicide. It is therefore evident that Denmert is a potent selective inhibitor of the demethylation at the C–14 position in ergosterol biosynthesis.

The fungicide, triarimol, was also found to exhibit the same effect on sterol biosynthesis as Denmert.     

The Biosynthesis of a Novel Nicotine Alkaloid in the Trichomes of Nicotiana stocktonii

N-Hydroxyacylnornicotine, newly discovered from fresh plant tissue, was found entirely in the trichome exudate produced at the epidermis of the aerial part of Nicotiana stocktonii. Nicotine and nornicotine, but not N-hydroxyacylnornicotine, were present inside of the trichomes as well as other internal parts of the plant. Only nicotine was found in bleeding sap squeezed from cut roots or stems. Feeding of leaves with 2′-¹⁴C-labeled nicotine primarily yielded labeled nicotine, nornicotine, and N-hydroxyacylnornicotine. When similarly labeled nornicotine was fed to leaves as a precursor, a labeled N-hydroxyacylnornicotine was obtained, with a higher specific activity than with the [2′-¹⁴C]nicotine feeding. Based on these results, a synthesis route is suggested where nicotine is converted in the leaf to nornicotine, followed by trichome conversion of nornicotine to N-hydroxyacylnornicotine, and rapid secretion of this product.     

Metabolism of [2-3H]- and [6-3H]-nicotinic acid in intact Nicotiana tabacum plants

Earlier observations of Dawson on the relative incorporation of [2-³H]- and [6-³H]-nicotinic acid into nicotine have been confirmed in intact Nicotiana tabacum plants. All the tritium in the nicotine derived from [2-³H]-nicotinic acid was located at C-2 of the pyridine ring. However the radioactive nicotine derived from [6-³H]-nicotinic acid was not labelled specifically at C-6 with tritium. By carrying out feeding experiments with [6-¹⁴-C, 2-³H]- and [6-¹⁴C, ³H]-nicotinic acids, it was established that there was very little loss of tritium from C-2 and C-6 of nicotinic acid during 5 days of metabolism in the tobacco plant.     

N′-isopropylnornicotine: Its formation from nicotine in aged leaves of Nicotiana tabacum

An aqueous solution of nicotine-[2′-¹⁴C] was painted on the leaves of 4-month-old tobacco plants (Nicotiana tabacum) which were harvested 3 weeks later. This tracer was similarly applied to excised tobacco leaves which were allowed to dry in air for 4 weeks. The alkaloids, were extracted with the addition of N′-isopropylnornicotine, a compound which has been previously isolated from air-cured tobacco. Radioactive nicotine and nornicotine were isolated from the intact plants with only minute activity in the N′-isopropylnornicotine. All three of these alkaloids were radioactive from the air-cured leaves, and degradation of the labelled N'-isopropylnornicotine indicated that all the activity was located at the C-2′ position. A higher level of activity was found in N′-isopropylnornicotine which was obtained from excised leaves which were fed the nicotine- [2′- ¹⁴C] in aqueous acetone, and were treated on subsequent days with aqueous acetone. These results are consistent with the hypothesis that N′-isopropylnornicotine is produced in the curing of tobacco leaves by reaction of nornicotine (formed by the demethylation of nicotine) with acetoacetate, followed by decarboxylation and reduction. The ¹³C NMR chemical shifts of the methyl groups of N′-isopropylnornicotine and related 1-isopropylpyrrolidines which have chirality at the α-position of the pyrrolidine ring, are significantly different (up to 7.5 ppm).     

The Biosynthesis of Some Isothiocyanates and Oxazolidinethiones in Rape (Brassica campestris L.)

The incorporation of the radioactivity from acetate-1-¹⁴C, acetate-2-¹⁴C, dl-methionine-1-¹⁴C, dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, dl-allyl-glycine-2-¹⁴C, and dl-2-amino-5-hydroxyvalerate-2-¹⁴C into the aglycones of progoitrin, gluconapin, and glucobrassicanapin of maturing rape plants (Brassica campestris L.) was investigated. Radioactivity from dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, and acetate-2-¹⁴C were incorporated into the 3 major thioglucosides. The other organic compounds were poorly incorporated except for dl-allylglycine-2-¹⁴C into glucobrassicanapin. The results obtained suggest that the rape plant can synthesize amino acids by the condensation of acetate (as acetyl CoA) to α-keto acids to yield a homologue of the original amino acid. These newly formed amino acids are then employed to synthesize the 3 major thioglucosides.     

Studies on the Chemical Regulation of Alkaloid Biosynthesis in Tobacco Plants

Effects of helminthosporol and helminthosporic acid (H-acid) on nicotine biosynthesis in Nicotiana were examined. By the addition of these compounds into nutrient solution, nicotine formation in decapitated and intact plants of Nicotiana tabacum was decreased. In sterile culture of excised roots of Nicotiana rustica, H-acid reduced the yield of nicotine exerting no effect on growth rate. Incorporation of l-glutamic acid-U-¹⁴C and dl-ornithine-2-¹⁴C into nicotine in the roots was decreased by the addition of H-acid, and that of nicotinic acid-³H(G) was not influenced. Since H-acid did not enhance destruction of nicotine-³H exogenously added, it is probable that the decrease of nicotine yield by the acid in the root culture is due to reduction in the production rate of the alkaloid.     

The effect of methionine on the metabolism of serine and glycine in vitamin B-12-deficient rats

The effect of methionine supplementation on glycine and serine metabolism was studied in vitamin B-12-deficient rats which received only 0.2% methionine in the diet. In the perfused liver, incorporation of the C-2 of glycine to the C-3 of serine was increased by addition of methionine to the perfusate. The oxidation of [1-¹⁴C]glycine to ¹⁴CO₂ was however depressed. Unlike methionine, glycine did not have any significant effect on the liver folate coenzyme distribution. Oxidation of [3-¹⁴C]serine to ¹⁴CO₂ both in vivo and in perfused liver was increased by methionine. A major portion of the C-3 radioactivity however was recovered in glucose. Data presented indicate that the rate of oxidation of [2-¹⁴C]histidine to ¹⁴CO₂ is more sensitive indicator of folate deficiency than the rate of oxidation of [3-¹⁴C] serine to ¹⁴CO₂ although both are presumably tetrahydrofolate dependent.     

Lipoic acid metabolism in the rat

dl-[1,6-¹⁴C]Lipoic acid was administered by intraperitoneal injection to rats at the level of 0.5 mg/100 g body weight. Approximately 56% of the radioactivity was recovered in the urine. When acidified and extracted with benzene, 92% of the radioactivity remained in the aqueous phase. Gel-filtration and paper chromatography were used to identify three of the compounds in the benzene extract as lipoic, bisnorlipoic and tetranorlipoic acids. In addition, a keto compound appears to be present. The aqueous phase contained several radioactive components separable by ion-exchange and paper chromatographies. Two of these compounds were identified as lipoate and β-hydroxybisnorlipoate. No evidence for oxidation of the dithiolane ring of lipoic acid was observed. dl-[7,8-¹⁴C]Lipoic acid was administered to rats under the same conditions. The urine contained 81% of the radioactivity, 72% of which remained in the aqueous phase and 28% was extracted into benzene. In contrast to over 30% of the label from dl-(1,6-¹⁴C] lipoate being expired as ¹⁴CO₂, a negligible amount of ¹⁴CO₂ was produced by rats injected with dl-[7,8-¹⁴C]lipoate. The catabolites identified were the same as those found using the 1,6-labeled lipoate. Another dithiolane-intact compound was also isolated. It appears that the rat, similar to Pseudomonas putida LP, metabolizes lipoate mainly via β-oxidation of the valeric acid side chain.