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 cholesteryl palmitate by rat brain in vitro; formation of cholesterol epoxides and cholestane-3beta,5alpha,6beta-triol

Incubation of [4-(14)C]cholesteryl palmitate with the 12,000 g supernatant fraction of adult rat brain fortified with an NADPH-generating system and beta-mercaptoethylamine resulted in formation (2-5%) of more polar metabolites characterized as a mixture of cholesterol-5,6-epoxides. Under extended incubation conditions, cholestane-3beta-5alpha-6beta-triol was isolated as the major end product of the incubations. Free [4-(14)C]cholesterol incubated under similar conditions was not oxidized, whereas oxidation of [4-(14)C]cholesteryl palmitate appeared to be dependent upon hydrolysis of the ester by the rat brain microsomal subcellular fraction. Elimination of the NADPH-generating system or the addition of EDTA to the incubation mixture inhibited epoxide formation, suggesting that the products are derived from an NADPH-dependent enzymatic lipoperoxidation mechanism. The in vitro conversion of [4-(14)C]cholesterol-5alpha,6alpha-epoxide to cholestane-3beta,5alpha,6beta-triol was also demonstrated in rat brain subcellular fractions in the absence of added cofactors.     

Biosynthesis of sterols and triterpenes in Pelargonium hortorum

Although all parts of the geranium plant (Pelargonium hortorum) are capable of synthesizing sterols and triterpenes and their esters in vitro from mevalonic acid-[2-¹⁴C], the aerial portions are more active than other tissues. All plant parts were shown to incorporate mevalonic acid-[2-¹⁴C] into isoprenoids for at least 3 days. The leaves and petioles had the greatest incorporation on a wet weight basis. Chopped preparations showed comparable incorporations of mevalonate whereby rootlets incorporated about one half as much as most parts; the flower petals incorporated five times the average amount. In leaves the principal sterol synthesized was sitosterol. Metabolic studies with isolated leaves indicated a fairly rapid conversion of free tetracyclic triterpenes to 4-desmethyl-sterols, while β-amyrin was synthesized at a different rate than α-amyrin. Esterified tetracyclic triterpenes exhibited only a slight amount of conversion to 4-desmethylsterols.     

Ascorbic acid metabolism in geranium and grape

l-Ascorbic acid-[UL-¹⁴C] has been used to follow the appearance of ¹⁴C-labeled oxalic acid and tartaric acid as metabolic products of oxidative cleavage of ascorbic acid in geranium apices (Pelargonium crispum). The enantiomeric specificity of ascorbic acid metabolism was established in geranium by comparing the incorporation of d- and l-ascorbic acid-[6-¹⁴C] in the presence of l-ascorbic acid-[4-³H]. l-Ascorbic acid-[4-³H] has been used to demonstrate the retention of ³H during biosynthesis of l-(+)-tartaric acid in the geranium and its exchange with water during biosynthesis of l-( +)-tartaric acid in the grape.     

Regulation of ketone body production from (14C)palmitate in rat liver mitochondria: effects of cyclic nucleotides and unlabeled fatty acids.

N⁶′, O²′-dibutyryl adenosine 3′, 5′-cyclic monophosphoric acid, but not other cyclic nucleotides stimulates [¹⁴C]ketone body production from [¹⁴C]palmitate in isolated rat liver mitochondria. Butyrate alone, as well as unlabeled acetate, octanoate and palmitate had similar effects. This redistribution of the oxidative products of [¹⁴C]palmitate can best be explained by exceeding the capacity of the Krebs cycle and/or changes in the acetyl coenzyme A/coenzyme A ratio. In contrast to [¹⁴C]palmitate, [¹⁴C]octanoate oxidation to [¹⁴C]O₂ and [¹⁴C]ketone bodies was inhibited by the addition of unlabeled fatty acids. This suggests that an additional mechanism by which unlabeled fatty acids may stimulate [¹⁴C]ketone body production is by enhancing the carnitine-dependent transport of [¹⁴C]palmitate into mitochondria.     

Monuron metabolism in excised gossypium hirsutum leaves: Aryl hydroxylation and conjugation of 4-chlorophenylurea*

A new glycoside was isolated as a minor metabolite in excised cotton leaves treated with either [carbonyl-¹⁴C] or [ring-¹⁴C] 3-(4-chlorophenyl)-1-methylurea and 4-chlorophenylurea. The aglycone from β-glucosidase or hesperidinase hydrolysis was identified as 4-chloro-2-hydroxyphenylurea by TLC, radioisotope dilution and MS.     

Comparison of oxidative metabolism in starved, fat-fed and carbohydrate-fed rats

1. Rats were starved for 48hr. or fed for 1 week on a high-fat or a high-carbohydrate diet. The effects of these dietary alterations on the rate of production of ¹⁴CO₂ from trace amounts of [U-¹⁴C]glucose, [1-¹⁴C]palmitate or [1-¹⁴C]acetate administered intravenously were studied. 2. The oxidation of [¹⁴C]glucose was most rapid in the carbohydrate-fed condition and was decreased significantly and to the same extent after starvation and after feeding with fat. 3. Under all dietary regimes studied the maximum rate of elimination of ¹⁴CO₂ from [¹⁴C]palmitate occurred within a few minutes after injection, but considerably more was oxidized after starvation and feeding with fat than after feeding with carbohydrate. 4. Alterations in diet had no effect on the oxidation and high recovery of administered [¹⁴C]acetate as ¹⁴CO₂. 5. Graphical analysis showed the presence of several exponential components in the ¹⁴CO₂-elimination curves. 6. In all studies a marked similarity in oxidative pattern was noted between the starved and the fat-fed rat.     

Lipid biosynthesis in warm- and cold-acclimatized sea anemones, Metridium senile (L.)

Sea anemones, Metridium senile (L.), naturally acclimatized to warm (18°C) and cold (0°C) conditions, were exposed to either [2-¹⁴C]acetate, [16-¹⁴C]palmitate, or [4-¹⁴C]cholesterol for periods up to 24 h. Isotope incorporation into triglyceride (TG), wax esters (WE), and polar lipid (PL) was recorded. Compared to warm-acclimatized groups, incorporation of [2-¹⁴C]acetate into WE of cold anemones was dramatically reduced, while TG incorporation remained at about the same levels. Highest values were recorded for PL in both groups. Using radiolabeled palmitate, incorporation values for WE were very low in both acclimatization groups though TG uptake remained comparatively high. Also noteworthy was a significant decrease in PL activity in cold anemones. Fatty acid analysis of total lipid, wax ester, triglyceride and phospholipid fractions showed a general shift towards increased chain length and unsaturation in cold-acclimatized anemones.     

Die quantitative verteilung der sterine und sterinderivate in organen von Solanum dulcamara

The following sterols were found in the roots, stems, leaves, unripe and ripe fruits of Solanum dulcamara: cholesterol, sitosterol, stigmasterol, campesterol, brassicasterol, isofucosterol and 24-methylenecholesterol. The most abundant components are cholesterol, sitosterol and stigmasterol (77–84%). In all parts of the plant the sterols are present in the free form and as esters, glycosides and acylated glycosides. The total sterol content and the content of combined forms were determined photometrically. In the leaves 58% of the sterols were found in the form of glycoside (26%), acylated glycoside (29%) and ester (2%). In the roots only 25% of the sterol were found in combined form. In the other organs the ratio of free and combined sterols was intermediate. In all cases, the ester fraction was the least.     

Sink to source translocation in soybean

The possibility that phloem loading may occur in the reproductive sink tissues of soybeans (Glycine max Merr. cv Chippewa 64) was examined. When [¹⁴C]sucrose was applied to seed coat tissues from which the developing embryo had been surgically removed, 0.1% to 0.5% of the radioactivity was translocated to the vegetative plant parts. This sink to source translocation was largely unaffected by destroying a band of phloem with steam treatment on the stem above and below the labeled pod. The same steam treatment, however, completely abolished translocation of [¹⁴C]sucrose between mature leaves and developing fruits. These results indicate that the movement of nutrients from developing seed coats to the vegetative plant parts occur in the xylem and that phloem loading does not occur in this sink tissue.     

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium

The metabolic fate of l-[4-¹⁴C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-¹⁴C]ascorbic acid and l-[6-¹⁴C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-¹⁴C]ascorbic acid and l-[1-¹⁴C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-¹⁴C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-¹⁴C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.     

Transport and Metabolism of 1-Aminocyclopropane-1-carboxylic Acid in Sunflower (Helianthus annuus L.) Seedlings

Transport and metabolism of [2,3-¹⁴C] 1-aminocyclopropane-1-carboxylic acid (ACC) from roots to shoots in 4-day-old sunflower (Helianthus annuus L.) seedlings were studied. [¹⁴C]ACC was detected in, and ¹⁴C₂H₄ was evolved from, shoots 0.5 hours after [¹⁴C]ACC was supplied to roots. Ethylene emanation from the shoots returned to normal levels after 6 hours. The roots showed a similar pattern, although at 24 hours ethylene emanation was still slightly higher than in those plants that did not receive ACC. [¹⁴C]N-malonyl-ACC (MACC) was detected in both tissues at all times sampled. [¹⁴C]MACC levels surpassed [¹⁴C]ACC levels in the shoot at 2 hours, whereas [¹⁴C]MACC levels in the root remained below [¹⁴C]ACC levels until 6 hours, after which they were higher. Thin-layer chromatography analysis identified [¹⁴C] ACC in 1-hour shoot extracts, and [¹⁴C]MACC was identified in root tissues at 1 and 12 hours after treatment. [¹⁴C]ACC and [¹⁴C] MACC in the xylem sap of treated seedlings were identified by thin-layer chromatography. Xylem transport of [¹⁴C]ACC in treated seedlings, and transport of ACC in untreated seedlings, was confirmed by gas chromatography-mass spectrometry. Some evidence for the presence of [¹⁴C]MACC in xylem sap in [¹⁴C]ACC-treated seedlings is presented. A substantial amount of radioactivity in both ACC and MACC fractions was detected leaking from the roots over 24 hours. A second radiolabeled volatile compound was trapped in a CO₂-trapping solution but not in mercuric perchlorate. Levels of this compound were highest after the peak of ACC levels and before peak MACC levels in both tissues, suggesting that an alternate pathway of ACC metabolism was operating in this system.     

Lipid biosynthesis in developing and germinating soybean cotyledons: The formation of palmitate and stearate by chopped tissue and supernatant preparations

Chopped tissue from developing soybean cotyledons incorporated [1-¹⁴C]acetate into palmitate, stearate, oleate, and linoleate, but with germinating cotyledons much less [1-¹⁴C]acetate was incorporated and the principal labeled products were palmitate, stearate, and oleate. When supernatant fractions from developing cotyledons were incubated with [1-¹⁴C]acetate or [2-¹⁴C]malonate the principal labeled products were palmitate and stearate. Supernatant fractions from germinating seed incorporated [2-¹⁴C]malonate into palmitate and also into short chain fatty acids including decanoate, laurate, and myristate. Supernatants from developing cotyledons required acyl carrier protein (ACP), ATP, CoA, and reduced pyridine nucleotides for maximal rates of incorporation of either [1-¹⁴C]acetate or [2-¹⁴C]malonate into palmitate and stearate. The de novo fatty acid synthetase which converts acetyl- and malonyl-ACP's to palmityl ACP was active in supernatant fractions from both young and old developing cotyledons. The elongation system, converting palmityl ACP to stearyl ACP, was more active in supernatants from younger than from older developing cotyledons. In experiments with chopped tissue the elongation system appeared equally active throughout the development process. These results are consistent with the view that the de novo and elongation systems are separate entities and that the elongation system in older cotyledons is less stable to the methods used to prepare supernatant fractions.     

Phospholipid metabolism in roots and microsomes of sunflower seedlings: Inhibition of choline phosphotransferase activity by boron

The action of boron on phospholipid composition and synthesis in roots and microsomes from sunflower seedlings has been studied. The fatty acid composition and relative amounts of individual molecular species of phospholipids in roots and microsomes were very similar. In both the content of phospholipids was decreased and the relative levels of their component fatty acids changed by treatment with 50 ppm of boron. This concentration of boron in the culture medium was found to inhibit the in vivo [1-^14C] acetate incorporation into root lipids and that of [Me-¹⁴C] choline into phosphatidylcholine of root microsomes. Cytidine-5-diphospho (CDP)-[Me-¹⁴C] choline incorporation into phosphatidylcholine of isolated microsomes was also inhibited by 50 ppm of boron when present in the growth medium of seedlings. These results indicate that the decrease in phosphatidylcholine labelling from [¹⁴C] choline observed when root microsomes were treated with boron would be caused by a decrease in CDP-choline phosphotransferase activity.     

Measuring plasma fatty acid oxidation with intravenous bolus injection of 3H- and 14C-fatty acid

Accurate measures of plasma FA oxidation can improve our understanding of diseases characterized by impaired FA oxidation. We describe and compare the 24 h time-courses of FA oxidation using bolus injections of [1-¹⁴C]palmitate versus [9,10-³H]palmitate under postabsorptive, postprandial, and walking conditions. Fifty-one men and 95 premenopausal women participated in one condition (postabsorptive, postprandial, or walking), one tracer (¹⁴C- or ³H-labeled), and an acetate or palmitate study. Groups were matched for sex, age, and body mass index (BMI). At 24 h, cumulative [³H]acetate recovery as ³H₂O was 80 ± 6%, 78 ± 2%, and 81 ± 6% in the postabsorptive, postprandial, and walking conditions, respectively (not significant). Model-predicted maximum [1-¹⁴C]acetate recovery as expired ¹⁴CO₂ was 59 ± 12%, 52 ± 8%, and 65 ± 10% in the postabsorptive, postprandial, and walking condition, respectively (one way ANOVA, P = 0.12). When corrected with the corresponding acetate recovery factors, 24 h time-courses of FFA oxidation were similar between [1-¹⁴C]palmitate and [9,10-³H]palmitate in all three conditions. In contrast to previous meal ingestion studies, an acetate-hydrogen recovery factor was needed to achieve comparable oxidation rates using an intravenous bolus of [³H]palmitate. In conclusion, intravenous boluses of [9,10-³H]palmitate versus [1-¹⁴C]palmitate gave similar estimates of 24 h cumulative FFA oxidation in age-, sex- and BMI-matched individuals.     

The importance of glyoxylate in amino acid biosynthesis in plants

1. [¹⁴C₂]Glyoxylate was rapidly metabolized by carrot storage tissues, pea leaves, pea cotyledons, sunflower cotyledons, corn coleoptiles, corn roots and pea roots. In many tissues over 70% of the supplied [¹⁴C₂]glyoxylate was utilized during the 6hr. experimental periods. 2. In all tissues, the chief products of [¹⁴C₂]-glyoxylate metabolism were carbon dioxide, glycine and serine. In several of the tissues, there was also a considerable incorporation of the label into the organic acids, particularly into glycollate. 3. Degradations of the labelled serine produced during [¹⁴C₂]glyoxylate metabolism showed that glyoxylate carbon was incorporated into all three positions of the serine molecule. 4. The results are interpreted as indicating that glyoxylate is utilized by the tissues by pathways involving transamination, transmethylation, reduction and oxidative decarboxylation of the supplied glyoxylate.     

The utilisation of fatty-acid substrates in triacylglycerol biosynthesis by tissue-slices of developing safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) cotyledons

Developing cotyledons of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.) readily utilised exogenously supplied ¹⁴C-labelled fatty-acid substrates for the synthesis of triacylglycerols. The other major radioactive lipids were phosphatidylcholine and diacylglycerol. In safflower cotyledons, [¹⁴C]oleate was rapidly transferred to position 2 of sn-phosphatidylcholine and concomitant with this was the appearance of radioactive linoleate. The linoleate was further utilised in the synthesis of diacyl- and triacyl-glycerol via the reactions of the so-called Kennedy pathway. Supplying [¹⁴C]linoleate, however, resulted in a more rapid labelling of the diacylglycerols than from [¹⁴C]oleate. In contrast, sunflower cotyledons readily utilised both labelled acyl substrates for rapid diacylglycerol formation as well as incorporation into position 2 of sn-phosphatidylcholine. In both species, however, [¹⁴C]palmitate largely entered sn-phosphatidylcholine at position 1 during triacylglycerol synthesis. The results support our previous in-vitro observations with isolated microsomal membrane preparations that (i) the entry of oleate into position 2 of sn-phosphatidylcholine, via acyl exchange, for desaturation to linoleate is of major importance in regulating the level of polyunsaturated fatty acids available for triacylglycerol formation and (ii) Palmitate is largely excluded from position 2 of sn-phosphatidylcholine and enters this phospholipid at position 1 probably via the equilibration with diacylglycerol. Specie differences appear to exist between safflower and sunflower in relation to the relative importance of acyl exchange and the interconversion of diacylglycerol with phosphatidylcholine as mechanisms for the entry of oleate into the phospholipid for desaturation.Abbreviations FW fresh weight - TLC thin-layer chromatography     

Axonal transport of phospholipids in rabbit optic pathway

The uptake of different labeled precursors, their incorporation into lipids, and transport along the rabbit optic pathway [ipsilateral retina and optic nerve (ON), and contralateral optic tract (OT), lateral geniculate body (LGB), and superior colliculus (SC)] were investigated. Albino rabbits were used. The following radioactive precursors, either combined or separately, dissolved in 50 l of saline containing 15% BSA, were injected into vitreous body: [2-³H]glycerol (50 Ci), [1-¹⁴C]palmitate (15 Ci), and [1-¹⁴C]linoleate (7.5 Ci). Animals were killed at different time intervals from 1 hr up to 24 days. The radioactivity of total lipids and of different phospholipid classes from total tissue was measured. One hour after the administration of precursors, the radioactivity into the retina was high and the incorporation of [³H]glycerol and [¹⁴C]palmitate increased until 12 hr and 24 hr, respectively. The incorporation of [¹⁴C]linoleate reached a maximum on the second day. The phospholipids of LGB and SC were intensively labeled after 4–8 hr, and their radioactivity increased up to the 10th day after injection, independent of the precursor employed. The results obtained indicate that the labeled hydrophilic and hydrophobic precursors used were actively incorporated into the retina. The phospholipids were later transported at a rapid rate along the optic pathway.A preliminary report of this study has been presented at the Satellite ISN Meeting, Istanbul, September 8–10, 1979.     

Source,sink and hormonal control of translocation in wheat

Summary An analysis of the pattern of movement of ¹⁴C-labelled flag leaf assimilates in wheat (Triticum aestivum l. c.v. Gabo) during grain development, indicated that the greater the requirement for assimilates by the ear the more rapid was the speed of movement of these through the peduncle to the ear and also the lower their concentration. Experiments with [¹⁴C] indoleacetic acid ([¹⁴C]IAA) suggested that auxin production by the grains was not responsible for the control of assimilate translocation through the peduncle. Limiting the supply of available assimilates by shading the lower parts of the plant, did not significantly alter the speed of movement of ¹⁴C-photosynthate through the peduncle, while severing half of the vascular tissue in the peduncle altered the pattern of movement of ¹⁴C to the ear and enhanced the speed of movement of ¹⁴C through the remaining functional conducting tissue. These results are discussed in relation to the mechanism of translocation.     

HORMONAL CONTROL OF TRANSLOCATION OF PHOTOSYNTHETICALLY ASSIMILATED 14C IN YOUNG SOYBEAN PLANTS

The normal supply of growth substances to a young soybean plant was altered by removing the plant's apical meristem and replacing this meristem with an aqueous solution of either indole-3-acetic acid (IAA), gibberellic acid (GA), or water. The length of each experiment was 1 hr. In the middle of it, ¹⁴CO₂ was administered to one of the primary leaves of the plant, and at the end distribution of ¹⁴C in various parts of the plant was determined. It was found that an addition of growth substances stimulated translocation in three different ways. Both IAA and GA increased the total amounts of sucrose-¹⁴C translocated, increased the rate of its translocation, and affected the distribution pattern of translocated sucrose throughout the plant. Experiments using IAA-¹⁴C have shown that the action of IAA is on the longitudinal translocation in the stem and not on the transfer of photosynthate from the mesophyll to the conducting tissues of the leaf.