The triterpenes are a large and diverse group of plant natural products that have important functions in plant protection and food quality, and a range of pharmaceutical and other applications. Like sterols, they are synthesised from mevalonate via the isoprenoid pathway, the two pathways diverging after 2,3-oxidosqualene. During triterpene synthesis 2,3-oxidosqualene is cyclised to one of a number of potential products, the most common of these being the pentacyclic triterpene beta-amyrin. Plants often produce complex mixtures of conjugated triterpene glycosides which may be derived from a single triterpene skeleton. The delineation, functional analysis and exploitation of triterpene pathways in plants therefore represent a substantial challenge. Here we have carried out high throughput screening to identify mutants of diploid oat (Avena strigosa) that are blocked in the early steps of triterpene synthesis. We also show that mutants that are affected in the first committed step in synthesis of beta-amyrin-derived triterpenes, and so are unable to cyclise 2,3-oxidosqualene to beta-amyrin (sad1 mutants), accumulate elevated levels of primary sterols. The major differences were in Delta-7-campesterol and Delta-7-avenasterol, which both increased several fold relative to wild-type levels. This is presumably due to accumulation of squalene and 2,3-oxidosqualene and consequent feedback into the sterol pathway, and is consistent with previous reports in which specific oxidosqualene cyclase inhibitors and elicitors of triterpene biosynthesis were shown to have inverse effects on the flux through the sterol and triterpene pathways.

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.

Four European Pulicaria species, P. odora, P. paludosa, P. sicula and P. vulgare, were analysed for their surface and vacuolar constituents for comparison with previous data obtained for P. dysenterica. Each species had a distinct flavonoid pattern with notable differences between leaf and inflorescence. 6-Hydroxyflavonols were the major lipophilic components in all of the species and tissues except in the leaves of P. paludosa and P. vulgare, where scutellarein 6-methyl ether was the main constituent. In the leaves of P. sicula a more unusual flavone, 6-hydroxyluteolin 5,6,7,3′,4′-pentamethyl ether, was a major component. Pulicaria odora was distinguished by the presence of a series of methylated 6-hydroxykaempferol derivatives including a 3,5,6,7,4′-pentamethyl ether. Quercetagetin hexamethyl ether occurred in both tissues of P. sicula together with the 3,7,3,4′-tetra methyl ether and other quercetagetin derivatives, which were 5-methylated. Quercetagetin 3,7,3′-methyl ether was present in all species except P. odora. Flavonol glucuronides were characteristic vacuolar constituents of all the taxa studied. Two rare glycosides, patuletin and 6-hydroxykaempferol 6-methyl ether 7-glucuronides were identified in the inflorescence of P. odora. Pulicaria vulgaris, a rare plant of southern England, had the vacuolar flavonoid profile most similar to the other more abundant British plant, P. dysenterica.

A method for measuring unlabelled Fe absorption has been investigated in a pilot study using a simple mathematical model. The metabolism of newly absorbed Fe can be approximated as a single-compartment model with the sampled compartment being the plasma pool. Five female volunteers (aged 30–55 years) were recruited to participate in the pilot study. After a 10 mg oral dose of unlabelled ferrous sulfate, the change in plasma Fe concentration over the following 6 h was used to estimate the quantity of absorbed Fe from the mathematical model. To assess the accuracy of the new technique, a 1 mg oral dose of 57Fe-labelled iron sulfate was given simultaneously with a 225 μg intravenous dose of 58Fe as iron citrate. The plasma appearance of the labelled Fe was used to estimate the absorption of the oral label from the traditional area under the curve method. There was no significant difference (P=0·61) between the geometric mean absorption of the unlabelled (19 (- 1 sd 12, +1 sd 28) %) and the labelled Fe (17 (- 1 sd 10, +1 sd 29) %). These initial results are encouraging, but further work needs to be undertaken with smaller doses, as typically found in meals. The effect of diurnal variation in serum Fe concentration on the estimation of unlabelled Fe absorption needs further assessment.

The major and structurally unique glucosinolate (GLS) in leaves of Eruca sativa L. (salad rocket) was identified as 4-mercaptobutyl GLS. Both 4-methylthiobutyl GLS and 4-methylsulfinylbutyl GLS were also present, but at lower concentrations. The 4-mercaptobutyl GLS was observed to oxidise under common GLS extraction conditions, generating a disulfide GLS that may be reduced efficiently by tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reform the parent molecule. The identities of 4-mercaptobutyl GLS and of the corresponding dimeric GLS were confirmed by LC/MS, MS/MS and NMR. Myrosinase treatment of an enriched GLS fraction or of the purified dimer GLS generated a mixture of unique bi-functional disulfides, including bis-(4-isothiocyanatobutyl) disulfide (previously identified elsewhere). TCEP reduction of the purified dimer, followed by myrosinase treatment, yielded only 4-mercaptobutyl ITC. GLS-derived volatiles generated by autolysis of fresh seedlings and true leaves were 4-mercaptobutyl ITC (from the newly identified GLS), 4-methylthiobutyl ITC (from 4-methylthiobutyl GLS) and 4-methylsulfinylbutyl ITC (from 4-methylsulfinyl-butyl GLS); no unusual bi-functional disulfides were found in fresh leaf autolysate. These results led to the conclusion that, in planta, the new GLS must be present as 4-mercaptobutyl GLS and not as the disulfide found after extraction and sample concentration. This new GLS and its isothiocyanate are likely to contribute to the unique odour and flavour of E. sativa.

Leaf surface extracts of Biebersteinia orphanidis have yielded a complex mixture of five flavones with the unusual 5,7-dihydroxy-6,8-dimethoxy A ring substitution pattern. They are acerosin, hymenoxin, nevadensin, sudachitin and 5,7,4'-trihydroxy-6,8-dimethoxyflavone. Also present at the leaf surface are gardenin B, luteolin, apigenin, acacetin and the coumarin umbelliferone. The internal leaf flavonoids include the 7-glucosides of apigenin, luteolin and tricetin, together with the 7-rutinosides of apigenin and luteolin. This profile differs from those of B. heterostemon and B. odora. It appears that B. orphanidis is as highly distinctive in its flavonoid pattern as it is phytogeographically. The data also confirm the conclusion of other studies, including rbcL and atpB gene sequence analysis, that Biebersteinia is completely unrelated to the Geraniaceae, where it was once placed.

Seven species of Tanacetum of different geographical origins were analysed in leaf and flower for their lipophilic and polar flavonoids and the results were compared with the patterns previously recorded in T. parthenium and T. vulgare. The lipophilic constituents are based generally on 6-hydroxykaempferol 3,6,4′-trimethyl ether and quercetagetin 3,6,3′-trimethyl ether, but up to 11 other surface flavonoids are present. Methyl ethers of scutellarein and 6-hydroxyluteolin are present in species with corymbose capitula. A rare carbomethoxyflavone reported earlier in T. microphyllum could not be detected. The dominant pattern of vacuolar flavonoids is based on apigenin and luteolin 7-glucuronides. 6-Hydroxyluteolin 7-glucoside, present in T. vulgare, also occurs in T. pseudoachillea. Chrysoeriol 7-glucuronide is present in T. parthenium, T. macrophyllum and T. corymbosum. By contrast, quercetin 7-glucuronide characterises T. parthenium, T. corymbosum and T. cinerariifolium. On the basis of combined data of lipophilic and polar constituents, it is clear that several structures are useful chemotaxonomic characters at the species level in a taxonomically complex genus.

A range of analogues of N-methylputrescine and tropinone were fed to transformed root cultures of Nicotiana rustica and/or a Brugmansia candida×aurea hybrid. These cultures were made by the transformation of the relevant plant species with Agrobacterium rhizogenes. A number of the metabolites, notably those showing a relatively modest alteration in the N-alkyl substituent, were metabolized in vivo to form homologues of the normal alkaloids biosynthesized by these roots. These products were identified by GC/MS and comparison with some synthetic reference materials. Analogues with major alterations in the size of the N-alkyl substituent were not metabolized at all. In the N. rustica cultures, the analogues fed at 1 mM significantly affected the profile of normal alkaloids, with up to a 4-fold diminution in nicotine being found in the presence of N-n-propylputrescine. The ratio between alkaloids of the pyrrolidine series and the piperideine series was also affected. In contrast, the presence of the analogues in the B. candida×aurea hybrid culture at 1 mM did not inhibit or substantially interfere with the accumulation of the normal spectrum of alkaloids. The potential for using these cultures to make complex novel products from simple precursors is discussed.

BACKGROUND:Nutritional iron deficiency in infants over 4 months of age is one of the most common deficiency disorders. Dietary iron is comprised of non-haem and haem iron, the latter being absorbed by a separate pathway and more efficiently than non-haem iron. Fortification of infant weaning foods is one of the strategies adopted for preventing iron deficiency and the aim of this project was to examine the potential use of haem iron concentrate as a fortificant. METHODS:Sixteen non-anaemic 6-month old infants were recruited and allocated to two groups of 8. Each infant consumed 2 meals/day of a commercial weaning food (100 g) for 7 consecutive days containing 40 mg ascorbic acid and 2.5 mg haem iron/100 g (Group 1) or the same quantity of iron as ferrous sulphate plus 40 mg ascorbic acid (Group 2). Bioavailability was assessed by chemical balance using carmine to mark the beginning and end of the faecal collection. The effect of haem iron concentrate (as a candidate for the factor in meat that enhances iron absorption) was examined by measuring its effect on 57Fe-labelled non-haem iron absorption. RESULTS:There was no difference in iron balance between the two groups. Mean iron retention was 3.5 (SD 2.1) mg/day in Group 1 (haem iron) and 3.0 (SD 2.4) mg/day in Group 2 (ferrous sulphate). Haem concentrate did not enhance the absorption of 57Fe-labelled non-haem iron, Group 1: 1710 (SD 11.1)%, Group 2: 28.4 (SD 17.7)%. CONCLUSIONS:Haem iron concentrate appears to be a highly bioavailable form of iron when added to infant weaning foods. This protein is not, however, responsible for the enhancing effect of animal protein on non-haem iron absorption.

The bioavailability of iron glycine added to a vegetable infant weaning food was compared with ferrous sulfate. Stable, isotopically labeled compounds (57Fe or 58Fe) were mixed into the midday meal (1.4 mg added Fe/serving) and fed to 9-mo-old infants on alternate days for 8 d. Bioavailability, expressed as a percentage of the dose consumed, was measured from isotopic enrichment of hemoglobin 14 d after the last test meal. There was no difference between iron glycine and ferrous sulfate (x+/-SEM): 9.0+/-0.7% and 9.9+/-0.8%, respectively. The effect of chelation was examined by measuring iron bioavailability of iron glycine and ferrous sulfate added to a high-phytate (310 mg/100 g) whole-grain cereal weaning food and comparing it with a lower-phytate (147 mg/100 g) vegetable food, as used in the first study. Both iron compounds had lower bioavailability from the high-phytate food, 5.2+/-0.5% for iron glycine and 3.8+/-0.9% for ferrous sulfate, than the lower-phytate food, 9.8+/-1.5% for iron glycine and 9.1+/-1.3% for ferrous sulfate. The results showed no significant difference in bioavailability between the two forms of iron when added to infant weaning foods, suggesting that the glycine complex was fully or partially dissociated in the gastrointestinal tract. It is concluded that chelation does not improve the bioavailability of iron in the presence of dietary inhibitors.

The rates (and extent) of appearance of glucose in arterialized plasma from an oral glucose load and from liver (RaO, RaH) can be estimated in humans using radioisotopes, but estimates vary among laboratories. We investigated the use of stable isotopes and undertook 22 primed intravenous infusions ofd-[6,6-2H2]glucose with an oral load includingd-[13C6]glucose in healthy humans. The effective glucose pool volume (VS) had a lower limit of 230 ml/kg body weight (cf. 130 ml/kg commonly assumed). This VSin Steele’s one-compartment model of glucose kinetics gave a systemic appearance from a 50-g oral glucose load per 70 kg body weight of 96 ± 3% of that ingested, which compared with a theoretical value of ∼95%. Mari’s two-compartment model gave 100 ± 3%. The two models gave practically identical RaOand RaHat each point in time and a plateau in the cumulative RaOwhen absorption was complete. Less than 3% of13C was recycled to [13C3]glucose, suggesting that recycling errors were practically negligible in this study. Causes of variation among laboratories are identified. We conclude that stable isotopes provide a reliable and safe alternative to radioactive isotopes in these studies.

Estimates of the spanchnic retention and appearance in the systemic circulation of orally administered glucose vary among laboratories even after recently identified sources of error have been accounted for [Livesey, G., P. D. G. Wilson, J. R. Dainty, J. C. Brown, R. M. Faulks, M. A. Roe, T. A. Newman, J. Eagles, F. A. Mellon, and R. Greenwood. Am. J. Physiol. 275 ( Endocrinol. Metab. 38): E717–E728, 1998]. We questioned whether, in healthy humans,d-glucose delivered intraluminally to the midjejunum appeared systemically as extensively as that delivered intraduodenally. Subjects were infused over a period of 90 min with 50 g of glucose in 1 liter of isotonic saline (incorporating 0.5 gd-[13C6]glucose) per 70 kg of body weight. Infusions were via enteral tubes terminating ∼15 and 100 cm postpylorus. The systemic appearance of glucose was monitored by means of a primed-continuous intravenous infusion ofd-[6,6-2H2]glucose. Whereas 98 ± 2% ( n = 7) of the duodenally infused glucose appeared in the systemic circulation, only 35 ± 9% ( n = 7) of midjejunally infused glucose did so, implying that 65 ± 9% was retained in the splanchnic bed. Either glucose was less efficiently absorbed at the midintestinal site or hepatic glucose sequestration was increased 10-fold, or both. The proximal intestine plays a key role in the delivery of glucose to the systemic circulation, and the distal intestine potentially delivers more glucose to the liver.

In a chemotaxonomic survey of 57 Pelargonium species, leaf exudate flavonoids were detected in 35% of the sample, mostly in trace amounts. However, chrysin and a related C-methylflavanone were identified as major leaf surface constituents of P. crispum, and a mixture of quercetin and kaempferol mono-, and di- and trimethyl ethers of P. quercifolium. In two other species, P. fulgidum and P. exstipulatum, methylated flavones were the only lipophilic flavonoids present. This is the first report of leaf surface flavonoids from the genus Pelargonium.

The use of rare earth elements as nonabsorbable fecal markers for studies of iron absorption from sources labeled extrinsically with stable isotopes was evaluated. On 3 successive days 13 healthy fasting adults were given different stable isotopes of iron with samarium, ytterbium, or dysprosium. On day 1, three meals were given with 57Fe (1 mg per meal) plus samarium (0.33 mg per meal); on day 2, identical meals (taken with a calcium supplement to reduce iron bioavailability) were given with equivalent amounts of 58Fe-labeled iron and ytterbium; on day 3, a well-absorbed reference dose of 54Fe (3 mg) was given with 1 mg Dy. A complete fecal collection was carried out for 5-9 d and each stool was analyzed for rare earth elements by inductively coupled plasma-mass spectrometry and iron isotopes by thermal ionization quadrupole mass spectrometry. Mean recovery of rare earth elements was 101%, indicating that they are totally unabsorbed. The excretory pattern of the iron isotopes and the rare earth elements was very similar; the correlation coefficients between samarium and 57Fe, ytterbium and 58Fe, and dysprosium and 54Fe were 0.992, 0.989, and 0.988, respectively (P < 0.001). Iron absorption was calculated as the difference between isotope dose and fecal excretion. Mean (+/-SEM) iron absorption was 16.7 +/- 2.4%, 4.3 +/- 1.6%, and 40.3 +/- 3.1% on days 1-3, respectively. Predicted values estimated from the first 4 d of pooled feces, using the rare earth element recovery data to produce corrected figures for unabsorbed isotope, were in close agreement: 19.1 +/- 2.1%, 4.6 +/- 1.7%, and 40.8 +/- 3.1%, respectively (P < 0.001). With the diet of medium iron bioavailability and with the highly bioavailable reference dose it was possible to predict iron absorption accurately from only one or two stools, provided that they were sufficiently enriched with isotope and a rare earth element.

The biosynthesis of the tropane alkaloids in transformed root cultures of Datura stramonium has been studied using sodium [1,2-C-13(2)]acetate, (R,S)-[2,3-C-13(2)]-1-(1-methyl-2-pyrrolidinyl)propane-2-one {(R,S)-[2',3'-C-13(2)]hygrine}, ethyl (R,S)-[1,2-C-13(2),2-C-14]-2-(1-methyl-2-pyrrolidinyl)acetate, and ethyl (R,S)-[2,3-C-13(2),3-C-14]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate. The incorporation of (R,S)-[2',3'-C-13(2)]hygrine into cuscohygrine and several other condensation products was high (15-40% specific incorporation), but label was not recovered in either tropine or tropine esters (hyoscyamine; 0.0 +/- 0.5% specific incorporation). None of the recovered alkaloids was labeled when ethyl (R,S) [1,2-C-13(2),2-C-14]-2-(1-methyl-2-pyrrolidinyl)acetate was fed to the cultures. In contrast, sodium [1,2-C-13(2)]acetate and ethyl (R,S)-[2,3-C-13(2),3-C-14]-4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate were incorporated into hyoscyamine (9 and 2% specific incorporation, respectively) and a number of other tropane alkaloids (up to 12% specific incorporation). These data provide further evidence that hygrine is not a direct precursor of tropane alkaloids. C-13-Label from acetate was incorporated symmetrically into the C-2 and C-4 positions of(-)-hyoscyamine, The evidence supports a pathway in which acetoacetate reacts via its C-4 position with N-methyl-Delta(1)-pyrrolinium salt to give 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoate. This intermediate favors cyclization to give 2-carboxytropinone, tropinone being formed by decarboxylation.