Chemistry of sulphur-bound pyrrolic metabolites in the blood of rats given different types of pyrrolizidine alkaloid.

Rats were injected with the pyrrolizidine alkaloids heliotrine, indicine, or anacrotine, and killed after 20 hr. Alkaloid metabolites conjugated to haemoglobin thiol groups were recovered in the form of pyrrolic monoethyl ethers, by treating blood samples with ethanolic silver nitrate under "buffered" conditions. Chemically prepared putative toxic metabolites of the alkaloids--dehydroheliotrine, dehydroindicine, and dehydroanacrotine--were also allowed to react in vitro with blood and with an immobilized thiol, thiol-Sepharose, and subsequently the S-conjugated pyrroles were again recovered as ethyl ethers. The recovered pyrrolic ethers were identified by comparing them with reference compounds prepared from ethanol and the dehydro-alkaloids, and the structures of the S-bound pyrroles were deduced. Blood from rats given the 9-monoester alkaloids heliotrine or indicine contained pyrrolic residues, S-bound at their 9-position. Anacrotine-treated rats yielded two diastereomeric 7-ethers, showing that dehydrocrotanecine 7-conjugates had been present in the blood. The products from alkaloid-treated rats were identical with those from blood or thiol-Sepharose treated with the corresponding dehydro-alkaloids in vitro. This supported the view that proximal metabolites leading to S-binding in vivo were the dehydro-derivatives of the alkaloids. In each case the thiols were attacked by the most reactive centre of the dehydro-alkaloid: the 9-ester in dehydroheliotrine and dehydroindicine, and the 7-ester in dehydroanacrotine. Accordingly, simple chemical reactions could account for the products formed in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute hepatotoxicity and pyrrolic metabolites in rats dosed with pyrrolizidine alkaloids

Relationships can be demonstrated between the acute hepatotoxicity of some pyrrolizidine alkaloids and the amounts of pyrrolic metabolites found in livers of rats given the alkaloids.     

Relation of structural features to pyrrolic metabolites in livers of rats given pyrrolizidine alkaloids and derivatives

Levels of pyrrolic metabolites have been measured in the livers of rats given some pyrrolizidine alkaloids and semisynthetic derivatives. Structural and chemical features favouring the formation of such metabolites have been defined. The most important of these were: steric hindrance or chemical properties giving resistance to ester hydrolysis; lipophilic character, allowing access to hepatic microsomal enzymes; a conformation favoring microsomal oxidation of the pyrroline ring in preference to N-oxidation. In addition, the presence of ester groups gave the resulting pyrroles high chemical reactivity, leading to tissue binding.Amounts of pyrroles bound to liver were very low when animals were given either highly water soluble pyrrolizidine derivatives, including non-esterified bases or more-lipophilic esters if these were easily hydrolysed. Compounds prone to hydrolysis gave increased pyrrole levels in rats pretreated to deplete their esterase activity. Whereas heliotridine-based alkaloids usually give more pyrrole than similar retronecine esters, heliotridine ditiglate gave less pyrrole than retronecine ditiglate because the former was more open to hydrolytic attack.Among the carboxylic diesters, the cyclic retronecine diesters, in which the pyrrolizidine nucleus is more exposed to oxidative metabolism, gave the highest pyrrole levels in rats.Liver pyrrole measurements are useful for studying relationships between molecular structure, metabolism and toxicity of pyrrolizidine derivatives. They can be used for screening alkaloids for potential toxicity and for assessing dose levels suitable for toxicity tests when limited material is available.     

Isolation of the pyrrolizidine alkaloid 1-epialexine from Castanospermum australe

The Australian leguminous tree Castanospermum australe contains the anti-viral glucose analogue indolizidine alkaloid castanospermine. As the result of a search for new bioactive carbohydrate-like compounds we now report the isolation of the novel polyhydroxylated pyrrolizidine alkaloid, 1-epialexine, from the leaves and stems of C. australe. 1-Epialexine is a weak inhibitor of β-mannosidase from Cellullomonas fimi.     

Selective uptake of pyrrolizidine N-oxides by cell suspension cultures from pyrrolizidine alkaloid producing plants

The N-oxides of pyrrolizidine alkaloids such as senecionine or monocrotaline are rapidly taken up and accumulated by cell suspension cultures obtained from plants known to produce pyrrolizidines, i.e. Senecio vernalis, vulgaris, viscosus (Asteraceae) and Symphytum officinale (Boraginaceae). The transport of the N-oxides into the cells is a specific and selective process. Other alkaloid N-oxides such as sparteine N-oxide are not taken up. Cell cultures from plant species which do not synthesize pyrrolizidine alkaloids are unable to accumulate pyrrolizidine N-oxides. The suitability of the pyrrolizidine N-oxides in alkaloid storage and accumulation is emphasized.     

Radioimmunoassay for biopterin in body fluids and tissues

Specific antibodies against l-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l-erythro-biopterin distinguished among l-erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l-erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-(l-erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with ¹²⁵I as the labeled ligand for the radioimmunoassay. BP-¹²⁵I-TYRA had similar binding affinity as the natural l-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP-¹²⁵I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H⁺ column was required for serum, cerebrospinal fluid, and brain tissues.     

Radioimmunoassay for neopterin in body fluids and tissues

Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absolute configuration of the pyrrolizidine alkaloid acetylgynuramine

The absolute configuration of the pyrrolizidine alkaloid acetylgynuramine was determined by X-ray diffraction to be (?)-(1aR,6bR,10R,11S)-9,14-dioxo-10-hydroxy-13-cis-ethylidene-11-methoxyacetyl-10-methyl-1a,2,3,6b-tetrahydro-5H-pyrrolizino-(1a,6b,6a,b,c)-1,8-dioxa-cyclododecane.     

Flavin-containing monooxygenase: a major detoxifying enzyme for the pyrrolizidine alkaloid senecionine in guinea pig tissues

Evidence based on optimal pH, thermal stability, and enzyme inhibition data suggests that the NADPH-dependent microsomal N-oxidation of the pyrrolizidine alkaloid senecionine is carried out largely by flavin-containing monooxygenase in guinea pig liver, lung, and kidney. In contrast, the hepatic microsomal conversion of senecionine to the pyrrole metabolite (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) is catalyzed largely by cytochrome P450. However, the rate of senecionine N-oxide formation (detoxication) far exceeded the rate of DHP formation (activation) in guinea pig liver microsomes over a range of pHs (pH 6.8 to 9.8). In guinea pig lung and kidney microsomes, N-oxide was the major metabolite formed from senecionine with little or no production of DHP. The high rate of detoxication coupled with the low level of activation of senecionine in liver, lung, and kidney may help explain the apparent resistance of the guinea pig to intoxication by senecionine and other pyrrolizidine alkaloids.     

Structure revision of emiline a pyrrolizidine alkaloid from Emilia flammea

The structure of emiline, a pyrrolizidine alkaloid isolated fromEmilia flammea has been revised from an 11-membered alkaloid to a 12-membered macrocyclic diester containing otonecine.     

Variation in pyrrolizidine alkaloid patterns of Senecio jacobaea

We studied the variation in pyrrolizidine alkaloid (PA) patterns of lab-grown vegetative plants of 11 European Senecio jacobaea populations. Plants were classified as jacobine, erucifoline, mixed or senecionine chemotypes based on presence and absence of the PAs jacobine or erucifoline. Due to the presence of jacobine, total PA concentration in jacobine chemotypes was higher than in erucifoline chemotypes. Both relative and absolute concentrations of individual PAs differed between half-sib and clonal families, which showed that variation in PA patterns had a genetic basis. Within most populations relative abundance of PAs varied considerably between individual plants. Most populations consisted either of the jacobine chemotype or of the erucifoline chemotype, sometimes in combination with mixed or senecionine chemotypes.     

Species difference in the urinary excretion of isatinecic acid from the pyrrolizidine alkaloid retrorsine

1. The urinary excretion of the metabolites, isatinecic acid and pyrrolic metabolites from the pyrrolizicline alkaloid retrorsine were lower in the resistant species, guinea-pigs, than the susceptible species, mice, hamsters and rats.2. The urinary N-oxides levels, however, were higher in guinea-pigs relative to mice, hamsters and rats.3. These results conform to the postulate that a common metabolic pathway exists between the formation of isatinecic acid and pyrrolic metabolites.4. The resistance of guinea-pigs to PA poisoning is attributed to the high metabolism of PAs to N-oxides combined with a corresponding low conversion to pyrrolic metabolites.     

The evolution of pyrrolizidine alkaloid biosynthesis and diversity in the Senecioneae

Pyrrolizidine alkaloids are characteristic secondary metabolites of the Asteraceae and some other plant families. They are especially numerous and diverse in the tribe Senecioneae and form a powerful defense mechanism against herbivores. Studies into the evolution of pyrrolizidine alkaloid biosynthesis using Senecio species have identified homospermidine synthase as the enzyme responsible for the synthesis of the first specific intermediate. These studies further indicated that the homospermidine synthase-encoding gene was recruited following gene duplication of deoxyhypusine synthase and that this occurred independently in several different angiosperm lineages. A review of published pyrrolizidine alkaloid data shows that the Senecioneae are characterized by a large qualitative and quantitative variation in pyrrolizidine alkaloid profiles and that these data demonstrate little phylogenetic signal. This suggests that although the first steps of this pathway are highly conserved, the diversification of secondarily derived pyrrolizidine alkaloids is extremely plastic.