The fate of [14C]thalidomide in the pregnant rabbit

1. The fate of [(14)C]thalidomide orally administered to pregnant rabbits at the beginning of the sensitive phase of pregnancy has been studied. 2. After the oral administration of [(14)C]thalidomide on the 192nd hour of pregnancy about 68% of the radioactivity appears in the urine and 22% in the faeces. 3. The urinary (14)C is made up as follows (% of dose): thalidomide (2); alpha-(o-carboxybenzamido)glutarimide (16); 2- and 4-phthalimidoglutaramic acids (11); 2-phthalimidoglutaric acid (0.2); 2- and 4-(o-carboxybenzamido)glutaramic acids and 2-(o-carboxybenzamido)-glutaric acid (29). 4. The plasma (14)C concentration is maximal at 12hr. after dosing and the radioactivity persists for more than 58hr. At 4hr. the main compound in the plasma is thalidomide, but its concentration steadily declines while the concentration of its hydrolysis products increases. 5. At 12, 24 and 58hr. after dosing radioactivity is present in the embryo and the maternal tissues examined. The (14)C concentration in the embryo is at nearly all times higher than that in the plasma, brain, skeletal muscle and fat but lower than that in the liver and kidney. 6. At 4hr. after dosing the mother on the tenth day of pregnancy the specific activities of the embryo and the yolk-sac fluid are similar. 7. Thalidomide is found in the embryo together with seven of its hydrolysis products for more than 24hr. after dosing. The accumulation of radioactivity in the embryo is due to retention of the polar hydrolysis products.     

5-Hydroxylevulinic acid,a new intermediate in the biosynthesis of protoanemonin

Administration of 5-hydroxy[1-¹⁴C]-and [4-¹⁴C]levulinic acid to Helleborus foetidus led to the isolation of [1-¹⁴C]- and [4-¹⁴C]protoanemonin, respectively. There was also incorporation of radioactivity into the four glucosides ranunculin, isoranunculin, ranuncoside and ranunculoside. Acid hydrolysis of radioactive ranuncoside gave labelled 5-hydroxylevulinic acid (HKV). A study of the incorporation of various ¹⁴C-labelled tracers into protoanemonin suggested that HKV is formed in higher plants by a new reduction of 2-ketoglutarate (2-KG) without free 4,5-dioxovalerate (DOVA) as an intermediate. A scheme for the biosynthesis of the antibiotic protoanemonin and its glucosidic precursors is proposed. It is shown that 5-(β-d-glucopyranosyloxy)levulinic acid could be the genuine precursor of all the compounds studied.     

Sites of synthesis of plasma proteins in the foetal rat

1. The foetal rat of 16 or more days incorporates ¹⁴C-labelled amino acids into all the demonstrable plasma protein fractions in vivo. 2. Slices of foetal rat liver incubated in vitro incorporate ¹⁴C-labelled amino acids into the main plasma protein fractions, including the foetal-specific `post-albumin'. 3. Slices of placenta are unable to incorporate ¹⁴C-labelled amino acids into plasma proteins in vitro. 4. Liver slices from maternal rats incubated in vitro incorporate ¹⁴C-labelled amino acids into plasma proteins. The presence of post-albumin cannot be demonstrated after incubation. 5. Liver slices from foetal rats, but not from adult rats, contain demonstrable amounts of haemoglobin into which ¹⁴C-labelled amino acids are incorporated.     

Isolation of glucosinolates and the identification of o-(α-l-rhamnopyranosyloxy)benzylglucosinolate from Reseda odorata

A method has been developed for the quantitative isolation of glucosinolates by ion-exchange chromatography and high voltage electrophoresis avoiding strongly alkaline and acidic conditions. The compounds were identified by ¹H and ¹³C NMR spectroscopy and through the products arising from enzymatic, acid and alkaline hydrolysis. The method is well suited for the isolation and identification of glucosinolates containing aglucone parts which produce non-volatile compounds on enzymatic hydrolysis. The method has been used in the isolation and identification of 2-hydroxy-2-methylpropylglucosinolate from Reseda alba, 2-hydroxy-2-phenylethylglucosinolate from R. luteola and a new glucosinolate, o-(α-l-rhamnopyranosyloxy)benzylglucosinolate, occurring in R. odorata. The glucosinolate content in different parts of this plant has been determined and the metabolism of glucosinolates is briefly discussed.     

Biosynthesis of glycosoaminoglycans by microsomal preparations from cultured mastocytoma cells

Neoplastic mast cells of mice (including long-established and newly derived lines) were grown in large-volume suspension cultures to provide enough cells for preparation of microsomal fractions. Microsomal preparations from P815Y and P815S cells synthesized ¹⁴C-labelled glycosaminoglycan when incubated with UDP-[¹⁴C]glucuronic acid and UDP-N-acetylgalactosamine. No significant amount of ¹⁴C-labelled glycosaminoglycan was formed when UDP-N-acetylglucosamine was substituted for the UDP-N-acetylgalactosamine. Microsomal preparations from X163 cells synthesized ¹⁴C-labelled glycosaminoglycan when incubated with UDP-[¹⁴C]glucuronic acid and either UDP-N-acetylgalactosamine or UDP-N-acetylglucosamine. The ¹⁴C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylgalactosamine was degradable by testicular hyaluronidase, indicating that it was chondroitin-like. The ¹⁴C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylglucosamine was not degradable by testicular hyaluronidase. Microsomal preparations from P815S cells were tested for sulphating activity by incubation with adenosine 3′-phosphate 5′-sulphatophosphate, as well as UDP-[¹⁴C]glucuronic acid, and UDP-N-acetylgalactosamine. The resulting newly synthesized polysaccharide was shown by chondroitinase ABC digestion to be 70% chondroitin 4-sulphate and 30% chondroitin. The molecular size of this newly synthesized glycosaminoglycan was determined by gel filtration to be larger than 40000 mol.wt. In general, the glycosaminoglycan-synthesizing ability of the microsomal preparations appeared to reflect glycosaminoglycan synthesis by the intact cells.     

Incorporation of chorismic acid and 4-aminobenzoic acid into the 4-hydroxyaniline moiety of N-(γ-L-glutamyl)-4-hydroxyaniline in Agaricusbisporus

Agaricus bisporus contains the unique aniline derivative, $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$. ¹⁴C-labelled chorismic acid was quantitatively incorporated into the 4-hydroxyaniline moiety of this aniline derivative, whereas ¹⁴C-labelled prephenic acid and anthranilic acid were not incorporated into 4-hydroxyaniline. These observations indicate the branch point of the biosynthetic route of 4-hydroxyaniline in the shikimic acid pathway to be chorismic acid. Moreover, 4-aminobenzoic acid proved to be an effective precursor of 4-hydroxyaniline.     

Mode of incorporation of precursors into alizarin (1,2-dihydroxy-9,10-anthraquinone)

The biosynthesis of alizarin (1,2-dihydroxy-9,10-anthraquinone) in Rubia tinctorum L. has been studied by tracer techniques. Specific incorporation of label from carboxyl-¹⁴C-d-shikimic acid, 2-¹⁴C-dl-glutamic acid and 5-¹⁴C-dl-Mevalonic acid suggests that these compounds provide the carbon skeleton of alizarin. Nonsymmetrical incorporation of label from carboxyl-¹⁴C-d-shikimic acid and 2-¹⁴C-dl-glutamic acid into alizarin indicates that the symmetrical 1,4-naphthoquinone is probably not an intermediate. Activity from o-(succinyl-2,3¹⁴C)-benzoic acid was found in the substituted benzene ring of alizarin. These data indicate that α-ketoglutaric acid or a derivative thereof combines with shikimic acid, chorismic acid or phrephenic acid to give o-succinylbenzoic acid which is then transformed to a nonsymmetrical intermediate γ,γ-Dimethylallylpyrophosphate is then attached, ring closure and further modification leading to alizarin.     

Biosynthesis of 1α,2α,3β-trihydroxy-p-menthane by Fusicoccum amygdali

Measurement of isotope ratios in 1α,2α,3β-trihydroxy-p-menthane, which has been biosynthesized in Fusicoccum amygdali from ³H- and ¹⁴C-labelled mevalonate and in its degradation product diosphenol indicates that: (a) four tritium atoms arising from [5-³H₂, 2-¹⁴C]MVA are retained, one more than suggested from the hydroxylation pattern, (b) menth-2-ene-1-ol is generated from an α-terpinyl cation through a 1,3-hydride shift and (c) trans-cleavage of an α-epoxide by hydrolysis gives 1α,2α,3β-trihydroxy-p-menthane.     

Attenuation of 2-methoxyethanol and methoxyacetic acid-induced digit malformations in mice by simple physiological compounds: Implications for the role of further metabolism of methoxyacetic acid in developmental toxicity

The ethylene glycol ether 2-methoxyethanol (ME) and its oxidation product methoxyacetic acid (MAA) are selective embryotoxins and equipotent as inducers of digit malformations when given by gavage to pregnant CrI:CD-1 ICR BR mice on gestation day 11. Earlier observations showed that the teratogenic effects were attenuated by delayed administrations of ethanol given at a time when all ME is already converted to MAA. That outcome suggested that acetate from ethanol catabolism might compete with methoxyacetate in biosynthetic reactions relevant to MAA-induced malformations. Furthermore, ¹⁴C derived from [1,2-¹⁴C]-ME or [1-¹⁴C]-MAA is incorporated into all marcromolecular fractions of the embryo, and ¹⁴C is exhaled by the dam in ¹⁴CO₂. Those data indicate that ¹⁴C derived from ¹⁴C-ME catabolism enters into many metabolic reactions. The present study examined acetate and other simple physiological compounds with close relationships to carbon and one-carbon moiety metabolic pathways for their ability to attenuate digit malformations upon concomitant dosing with ME. All of the agents examined reduced the teratogenic effect significantly with a potency rank order of formate ? acetate = glycine ? D-glucose. The common link for their efficacy may be the one-carbon moiety oxidation pathway that involves tetrahydrofolic acid as a catalyst of one-carbon transfer into purines and thymidylate. Carbon from all of the attenuators administered is incorporated into those bases and then into DNA. It appears as if methoxyacetate enters into biochemical reactions analogous to those of acetate. This speculation is supported by the metabolic fate of ¹⁴C from ¹⁴C-ME in dam and embryo. Based on the indirect evidence obtained with all of the simple compounds that attenuate the ME-induced digit malformations, we postulate that abnormal macromolecules are generated by anabolic reactions and that those products disrupt normal paw development.     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches has led to the identification of the structure of the persistently DNA-bound AAF moity. First, [3-³H, 9-¹⁴C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent—AAF moiety in DNA in vivo. The ration ³H/¹⁴C of this product had diminished to the same extent as 3-CH₃S-AAF resulting from the reaction of methionine with [3-³H, 9-¹⁴C]N-acetoxy-AAF.Secondly, reaction of [9-¹⁴C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally, by reacting N-OSO₃-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N²-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2′-³H]N-hydroxy-AAF.     

Excretion of Ribitol and Sucrose by Green Algae into the Culture Medium

Among 8 strains of algae grown with C¹⁴O₂ as a sole source of carbon, two species of Trebouxia produced appreciable amounts of two photosynthetic products in the culture medium. One of them was identified as sucrose by cochromatography and by acid hydrolysis. The other compound was identified as ribitol by paper chromatography, paper electrophoresis, periodic acid oxidation, recrystallization with authentic ribitol and finally by the enzymatic method with ribitol dehydrogenase.     

Biosynthesis of cyanogenic glucosides in butterflies and moths: Effective incorporation of 2-methylpropanenitrile and 2-methylbutanenitrile into linamarin and lotaustralin by Zygaena and Heliconius species (Lepidoptera)

¹⁴C-labelled 2-methylpropanenitrile and 2-methylbutanenitrile were administered to larvae and imagines of Heliconius melpomone and to larvae of Zygaena trifolii and the incorporation into the cyanogenic glucosides, linamarin and lotaustralin, was measured. Both species incorporated the precursors at all stages tested, at a high level of 15–72%, thereby indicating that the nitriles are probale intermediates in the lepidopteran biosynthesis of linamarin and lotaustralin from valine and isoleucine respectively.     

Degradation of phenylalanine and tyrosine by Sporobolomyces roseus

Ammonia-lyase activity for l-phenylalanine, m-hydroxyphenylalanine and l-tyrosine was demonstrated in cell-free extracts of Sporobolomyces roseus. Cultures of this organism converted dl-[ring-¹⁴C]phenylalanine and l-[U-¹⁴C]tyrosine into the corresponding cinnamic acid. Tracer studies showed that these compounds were further metabolized to [¹⁴C]protocatechuic acid. Benzoic acid and p-hydroxybenzoic acid were intermediates in this pathway. Washed cells of the organism readily utilized cinnamic acid, p-coumaric acid, caffeic acid, benzoic acid and p-hydroxybenzoic acid. Protocatechuic acid was the terminal aromatic compound formed during the metabolism of these compounds. The cells of S. roseus were able to convert m-coumaric acid into m-hydroxybenzoic acid, but the latter compound, which accumulated in the medium, was not further metabolized. 4-Hydroxycoumarin was identified as the product of o-coumaric acid metabolism by this organism.     

Metabolic fate of 3,4-dichloropropionanilide in plants: the metabolism of the propionic Acid moiety

3,4-Dichloropropionanilide-¹⁴C (propanil) labeled in either the C-1 or C-3 carbon atoms of the propionic acid moiety was applied to the roots of pea (Pisum sativum L.) and rice (Oryza sativa L.) plants in nutrient solution (0.1 mm-0.28 mm). Radioactivity was detected throughout the treated plants, but the greatest labeling was found in the roots. None of the products that contained aniline were radioactive, suggesting that the plants split the propionic acid moiety from propanil. The fate of the propionate moiety of propanil was determined by recovery of ¹⁴CO₂ from plants exposed to propanil-¹⁴C. The time-course of the ¹⁴CO₂ production demonstrated that the intact propionic acid was cleaved from the propanil and subsequently catabolized by the β-oxidation catabolic sequence. The appearance of radioactivity in the shoots was attributed to the incorporation of products of propionate metabolism. Both the susceptible pea plants and the tolerant rice plants converted a high percentage of the administered propanil-¹⁴C to ¹⁴CO₂.     

Dhurrin,the cyanogenic glucoside of Cercocarpus ledifolius

Dhurrin (2-β-d-glucopyranosyloxy-2-(4-hydroxy)phenyl-2S-acetonitrile) was isolated as a cyanogenic compound from Cercocarpus ledifolius and identified by its hydrolysis products, chromatographic properties and ¹H NMR spectroscopy.     

Biosynthesis of propyl cannabinoid acid and its biosynthetic relationship with pentyl and methyl cannabinoid acids

Biosynthesis of propyl cannabinoid acid has been determined by in vitro incubation with a crude enzyme solution from three strains of Cannabis sativa using ¹⁴C-labelled cannabinoid acid. Biosynthetic relationships between methyl, propyl and pentyl cannabinoid acids have been demonstrated.     

The 2-hydroxylation of trans-cinnamic acid by chloroplasts from Melilotus alba Desr

Chloroplasts isolated from sweetclover leaves contain an enzyme which converts trans-[3-¹⁴C]cinnamic acid to 2-hydroxy-trans-[3-¹⁴C]cinnamic (o-coumaric) acid. The identity of the product has been verified by recrystallization with unlabeled o-coumaric acid to constant specific activity, and by gas-liquid cochromatography of unlabeled o-coumaric acid and the radioactive product.The enzyme has an optimum of pH 7.0 and its activity can be enhanced ~ 4-fold by adding 4 mm glucose-6-phosphate to the reaction mixture. Light can replace glucose-6-phosphate, presumably as a source of reducing power required for the hydroxylation system. It was found that approximately 50% of the hydroxylase activity is bound to the lamellar membranes, from which it can be released by sonication.     

Metabolism of (±) 2-[14C]-abscisic acid in Lactuca sativa achenes

Achenes ofLactuca sativa L. cv. Grand Rapids were treated with (±) 2-[¹⁴C]-abscisic acid (ABA) at 10⁵ - or 2-10⁶ M for 6, 12, 24, 48 or 96 h in darkness at 24°C. They were then extracted in 80% ethanol. Two acidic diethyl ether phases which contained the free acids and the acids released after mild alkaline hydrolysis respectively, were analyzed as well as the radioactivity which remained in the final aqueous phase. For treatment durations between 6 and 96 h, the major part of the radioactivity was found in the free phase, in the form of ABA. For treatment durations up to 48 h, no radioactivity was detected at the Rf of phaseic acid or dihydrophaseic acid (free and hydrolysed phases). After 96 h culture on 10⁵ M ABA, dihydrophaseic acid was present, but only in very small quantities. Two ABA metabolites were detected. One was characterized as β-d -glucopyranosyl abscisate since its Rf was the same as that of an authentic sample in three different solvent systems and also since it released ABA on mild alkaline hydrolysis. It increased steadily with time and represented the main metabolite. The other metabolite found in the aqueous phase after mild alkaline hydrolysis and extraction with ether at pH 3 was a very polar compound, resistant to alkaline hydrolysis in the presence of concentrated ammonia and to methylation. It was, however, metabolized by apple embryo, yielding essentially dihydrophaseic acid and an ester which released dihydrophaseic acid on mild alkaline hydrolysis. These results indicate that under the conditions tried, the metabolism of [¹⁴C]-ABA by lettuce achenes leads almost exclusively to the formation of conjugates, oxidative metabolism of ABA being almost non-existent. Separate analysis of the integuments and of the endosperm plus embryo after culture of whole achenes for 48 h in the presence of 10⁵ M [¹⁴C]-ABA showed that ABA metabolism occurred only in the endospermembryo tissue.     

Inhibition by cerulenin of lipid synthesis in Crambe abyssinica tissues

Cerulenin is a potent inhibitor of fatty acid synthesis from ¹⁴C-labelled acetate in leaves and developing seeds of Crambe abyssinica. The antibiotic is equally inhibitory on the elongation of [1-¹⁴C]-oleic acid to erucie acid which is the major fatty acid of the seed. There is no significant inhibition of fatty acid desaturation in either tissue. Acylation of lipids is not a primary target of cerulenin's action.