In vivo conversion of [4-14C]sitosterol and [22,23-3H]sitosterol to stigmasterol in barley seedlings

Six-day-old barley seedlings were allowed to take up [4-¹⁴C]sitosterol and [22, 23-³H]sitosterol for 2.5 hr and the incorporation into the sterol fractions was determined after 0, 6, 12 and 24 hr. Sitosterol was readily incorporated into every sterol class. The ³H/¹⁴C ratio in the free forms dropped when compared with the ³H/¹⁴C ratio of the administered sitosterol. In the free sterol, radioactive stigmasterol, showing a ³H/¹⁴C ratio half that of the sitosterol ³H/¹⁴C ratio, was isolated and its radiochemical purity established by dilution with carrier material and crystallization to constant specific activity.     

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.     

ON THE ORIGIN OF THE ACETYL MOIETY OF ACETYLCHOLINE IN BRAIN STUDIED WITH A DIFFERENTIAL LABELLING TECHNIQUE USING 3H-14C-MIXED LABELLED GLUCOSE AND ACETATE

—Cortex slices of rat brain were incubated with glucose mixed-labelled with ³H and ¹⁴C in the 6-position and the ³H/¹⁴C ratios of lactate, acetate, citrate and acetylcholine were determined. The values obtained were: lactate 0·95, acetate 0·85, citrate 0·65 and acetylcholine 0·67 when expressed in relation to a glucose ³H/¹⁴C ratio of 1·00. When brain slices were incubated with [2-¹⁴C, 2-³H]acetate in the presence of unlabelled glucose, labelled acetylcholine was formed with a ³H/¹⁴C ratio not significantly different from the labelled substrate. The results indicate that citrate is a precursor to the acetyl moiety of acetylcholine.     

Biosynthesis of meteloidine from 3α-tigloyloxytropane in Datura innoxia

The administration of 3α-tigloyl-[1-¹⁴C]-oxytropane-[3β-³H] (³H/¹⁴C = 11·0 to Datura innoxia plants for 7 days led to the formation of radioactive meteloidine (³H/¹⁴C = 11·6). Degradation of the meteloidine indicated that the alkaloid was labeled specifically with ³H at C-3 of its teloidine moiety, and on the carbonyl group of its tigloyl residue with ¹⁴C. These results strongly favor the hypothesis that hydroxylation of tropine occurs after formation of its tigloyl ester.     

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.     

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′.     

Biosynthesis of nimbolide from [2-14C,(4R)4-3H1] mevalonic acid lactone in the leaves of Azadirachta indica

Nimbolide was biosynthesized from [2-¹⁴C, (4R)4-³H₁]mevalonic acid lactone in the leaves of Azadirachta indica. The nimbolide had a ³H:¹⁴C ratio of 3:5 which gives support to the suggestion of the involvement of a triterpenoid intermediate with a double bond at the Δ⁸⁽⁹)-position in the biosynthesis of nimbolide.     

The metabolism of l[3-3H]lactate by isolated hamster liver cells

The rate of tritium removal from l[3-³H]lactate by hamster liver cells is faster than the analytical rate of lactate utilization, or the rate of ¹⁴C disappearance from l[U-¹⁴C]lactate, with the result that the ³H/¹⁴C ratio in residual lactate from l-[U-¹⁴C,3-³H]lactate decreases. However, addition of low concentrations (0.1 to 1.0 mM) of l-cycloserine, a glutamate pyruvate transaminase inhibitor, nearly equalizes the rates of isotope utilization from l-[3-³H]lactate and l-[U-¹⁴C]lactate. The results suggest a very limited rate of recycling of phosphoenolpyruvate back to pyruvate during gluconeogenesis from lactate in fasted hamster liver cells.     

Production of diosgenin from yellow ginger (<Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> C. H. Wright) saponins by commercial cellulase

A commercial cellulase was first assessed to be effective in hydrolyzing glycosyl at the C-3 and C-26 positions in steroidal saponins from yellow ginger (Dioscorea zingiberensis C. H. Wright) to diosgenin, a very important chemical in the pharmaceutical industry. The effect of different parameters on enzyme hydrolysis was further investigated by systematically varying them. The highest yield was achieved when the hydrolysis ran at 55°C and pH 5.0 with an enzyme to substrate ratio of 15 × 10³ U/g. The biotransformed products identified using TLC and HPLC confirmed that the cellulase was capable of releasing diosgenin from steroidal saponins. Moreover, the biotransformation process was explored by LC-MS and LC-MS/MS analysis. Enzymatic hydrolysis together with 40 % of the original sulphuric acid used increased the diosgenin yield by 15.4 ± 2.7% than traditional method. Therefore, the commercial cellulase may serve as a promising tool for industrial diosgenin production and for further use in saponin modification.     

Incorporation of methionine-[methyl-2H3] and mevalonic acid-[2-14C,(4R)-4-3H1] into Phycomyces blakesleeanus sterols

Ergosterol, episterol, 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol and 24-methylene-24,25-dihydrolanosterol, isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. The ¹⁴C:³H atomic ratio of the following sterols isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁], was: ergosterol, 5:3; episterol, 5:4; ergosta-5,7,24(28)-trien-3β-ol, 5:3; 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol, 5:4; 24-methylene-24,25-dihydrolanosterol, 6:5; lanosterol, 6:5. The significance of these results in terms of ergosterol biosynthesis is discussed.     

Studies in phytosterol biosynthesis. Mechanism of biosynthesis of cycloartenol

1. The mechanism of cycloartenol biosynthesis in leaves of Solanum tuberosum was investigated with the use of [2-¹⁴C,(4R)-4-³H₁]mevalonic acid. 2. The ³H/¹⁴C atomic ratio in cycloartenol was 6:6, the same as that in squalene; this eliminates lanosterol as a possible biosynthetic precursor of cycloartenol, and indicates that a hydrogen migration from C-9 to C-8 occurs. 3. Chemical isomerization of the cycloartenol to lanosterol (³H/¹⁴C ratio 5:6) and parkeol (³H/¹⁴C ratio 6:6) confirms the hydrogen migration from C-9 to C-8. 4. Possible mechanisms for the biosynthesis of cycloartenol and parkeol are discussed. 5. The ³H/¹⁴C ratio for 24-methylenecycloartanol was 6:6, demonstrating that the hydrogen atom at C-24 is retained during alkylation of the cycloartenol side chain.     

Biosynthetic preparation of cardenolides from [1-14C]acetic acid by stem discs of the milkweed Asclepias curassavica

[¹⁴C]Calotropin (11.2 μCi/mmol) and uscharidin (14.1 μCi/mmol) were biosynthesized by stem discs of Asclepias curassavica incubated in a medium containing [1-¹⁴C]acetic acid. Relative isotope enrichment sites determined by ¹³C NMR spectroscopy of [¹³C]calotropin prepared by the same method were at C-23 (0.71 %), C-2′ (0.28 %) and C-4′ (0.21 %).     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

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.     

Biogenesis of indole compounds from D- and L-tryptophan in segments of etiolated seedlings of cabbage,maize and pea

The metabolism of D-and L-tryptophan-3-¹⁴C (Try-3-¹⁴C) was studied and compared for three different plant species, cabbage, maize and pea. Apical segments of the seedlings were incubated for 6 hours in solutions of L- or D-Try-3-¹⁴C (1·5 μc/ml) with the addition of chloramphenicol (10^?4g/ml) and then allowed to stand for another 20 hours in moist chambers. The methanolic extract of the tissues was analyzed radiochromatographically and by paper electrophoresis in combination with biological tests. Chloramphenicol in a concentration of 10^?4 g/ml had little influence on the growth of the segments, though the antibiotic slightly decreased the uptake of L-Try, it did not prevent the formation of IAA from L-Try. In the segments of cabbage the following metabolites were formed from L-Try-3-¹⁴C (accounting for 52% of the activity of the chromatographically separated extract): glucobrassicin (26·0%), neoglucobrassicin (3·6%), a spot corresponding according to its Rf to 3-indolylacetamide (IAAmide—10·9%), β-glucoside of 3-indolylacetic acid (IAGluc—3·3%) and traces of 3-indolylacetonitrile (IAN), IAA and indole-3-carboxylic acid (total 5%). In maize segments L-Try-3-¹⁴C (53·0%) was transformed to several unidentified hydrophilic substances, one of them possessing auxin activity (total amount 6·9%), IAGlue (9·3%) accompanied by a small amount of tryptamine, a spot corresponding according to its Rf to IAAmide (16·5%), IAA and another unidentified hydrophobic substance (4·1%). In pea segments L-Try-3-¹⁴C (66·7%) gave a zone corresponding according to its Rf to IAAmide (20·0%), a substance similar to IAGluc (10·5%) and also hydrophobic substances (3·1%) containing traces of IAA, which could be demonstrated only by bioassay. D-Try is metabolised in the three plants by the virtually exclusive formation of malonyltryptophan.     

Biosynthesis of hygrine from [5-14c]ornithine via a symmetrical intermediate in Nicandra physaloides1

Radioactive hygrine (2.2% incorporation) was isolated from Nicandra physaloides plants which had been fed Dl-[5-¹⁴C]ornithine. A systematic degradation of the hygrine yielded products whose activity was consistent with the pyrrolidine ring of this alkaloid being labeled equally at the C-2 and C-5 positions. The result does not agree with the previous work of O′Donovan and Keogh, whose publication is critically examined.     

Mechanism of alkylation at C-24 during ergosterol biosynthesis in Phycomyces blakesleeanus

Ergosterol isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃] contained two ²H atoms showing that one ²H atom is lost during transmethylation. Ergosterol isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁] had a ¹⁴C:³H atomic ratio of 5:3. Chemical degradation of 2,3-dimethylbutanal obtained by ozonolysis of the doubly-labelled ergosterol showed that the ³H atom originally at C-24 of lanosterol is transferred to C-25 of ergosterol during transmethylation. The mechanism of formation of the ergosterol side chain in P. blakesleeanus is presented.     

Formation of 3H2O from [2-3H]- and [4-3H]estradiol by rat uteri in vitro: Possible role of peroxidase

The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of ³H₂O from either [2-³H]- or [4-³H]estradiol. Hydrogen peroxide added to this system increased the yield of ³H₂O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H₂O₂ but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of ³H from [2-³H]- or [2,4,6,7-³H]- but not [4-³H]- or [6,7-³H]estradiol. It is proposed that the formation of ³H₂O from ³H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid.     

The stereochemistry of hydroxylation of the carotenoid lutein in Calendula officinalis

Excised, opening inflorescences of Calendula officinalis incorporated (3RS, 5R)- and (3RS, 5S)-[2-¹⁴C,5-³H₁]mevalonates into the carotenoid fraction. The ¹⁴C:³H ratios of lutein isolated from these tissues showed the hydrogen atom at C-3 of the β-ring is derived from the 5-pro-S position of mevalonate, while that at C-3 of the ε-ring is derived from the 5-pro-R position of mevalonate. Oxidation of lutein to monoketolutein showed that both hydrogen atoms at the C-15,15′ central double bond are derived from the 5-pro-R position of mevalonate.