C-nuclear magnetic resonance studies of the biosynthesis of 5-aminolevulinic acid destined for chlorophyll formation in dark-grown Scenedesmus obliquus

The ¹³C-nuclear magnetic resonance (NMR) spectra of chlorophyll a formed in dark-grown Scenedesmus obliquus (Turp.) Kützing in the presence of [1-¹³C]glutamate, [2-¹³C]- and [1-¹³C]glycineshowed that the ¹³C of glutamate was specifically incorporated into the eight-carbon atoms in the tetrapyrrole macrocycles derived from C-5 of 5-aminolevulinic acid (ALA), while the C-2 of glycine was only incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of chlorophyll a. No specific enrichment of these nine carbon atoms was observed in the spectrum of chlorophyll a formed in the presence of [1-¹³C]-glycine. These labeling patterns provide evidence for the operation of the C₅-pathway and against the operation of the ALA synthase pathway for chlorophyll formation in darkness.     

Biosynthesis of methyl branched hydrocarbons of the German cockroach Blattella germanica (L.) (orthoptera,blattellidae)

The precursors and directionality of synthesis of the methyl branched cuticular hydrocarbons and the female contact sex pheromone, 3,11-dimethyl-2-nonacosanone, of the German cockroach, Blattella germanica, were investigated by radiotracer and carbon-13 NMR techniques. The amino acids [G-³H]valine, [4,5-³H]isoleucine and [3,4-¹⁴C₂]methionine labeled the hydrocarbon fraction in a manner indicating that the carbon skeletons of all three amino acids serve as the methyl branch group donor. The incorporation of [1,4-¹⁴C₂]- and [2,3-¹⁴C₂]succinates into the hydrocarbon and acylglycerol/polar lipid fractions indicated that succinate also served as a precursor to methylmalonyl-CoA. Carbon-13 NMR analyses showed that [1-¹³C]propionate labeled the carbon adjacent to the tertiary carbon, and, for the 3,x-dimethylalkanes, that carbon-4 and not carbon-2 was enriched. [1-¹³C]Acetate labeled carbon-2 of these hydrocarbons. This indicates that the methyl branching groups of the 3,x-dimethylalkanes were inserted early in the chain elongation process. [3,4,5-¹³C₃]Valine labeled the methyl, tertiary and carbon adjacent to the tertiary carbon of the methyl branched alkanes. Thus, the methyl branched hydrocarbon was formed by the insertion of methylmalonyl units derived from propionate, isoleucine, valine, methionine and succinate early in chain elongation.     

On the biosynthesis of 5-hydroxybenzimidazolylcobamide (vitamin B12-factor III) in Methanosarcina barkeri

Exogenous 5-hydroxy-[2-¹⁴C]benzimidazole was transformed by Methanosarcina barkeri into 5-hydroxy-[2-¹⁴C]benzimidazolylcobamide. Thereby the endogenous biosynthesis of 5-hydroxybenzimidazole was completely blocked.Benzimidazole and 5,6-dimethylbenzimidazole were used by M. barkeri to form benzimidazolylcobamide respectively 5,6-dimethylbenzimidazolylcobamide (vitamin B₁₂), but in these cases the endogenous biosynthesis of factor III was not completely suppressed.With [2-¹⁴C]benzimidazole it was demonstrated that this base as well as the benzimidazolylcobamide formed thereof are no precursors in the biosynthesis of 5-hydroxybenzimidazolylcobamide.Glycine instead was found to be a building block for the biosynthesis of 5-hydroxybenzimidazole, since radioactivity from [1-¹⁴C] and [2-¹⁴C]glycine was incorporated, into the base moiety of factor III, but not into its corrin moiety. With [1-¹³C]glycine and ¹³C-NMR-spectroscopy it was shown that C-1 of glycine gets C-3a of 5-hydroxybenzimidazole.[1-¹³C]glycine also led to a single prominent signal in the ¹³C-NMR-spectrum of coenzyme F₄₂₀, this was assigned to C-10a.Thus C-1 of glycine was incorporated into the hydroxybenzene part of 5-hydroxybenzimidazole, whereas it was not incorporated into this part of coenzyme F₄₂₀, indicating that the hydroxybenzene part of these two compounds is not formed from a common intermediate. L-[U-¹⁴C]glutamate led to the exclusive labeling of the corrin ring of factor III, showing that the corrin precursor 5-aminolevulinic acid is formed by the C-5 pathway in M. barkeri.These experiments indicate that the biosynthesis of factor III in the archaebacterium M. barkeri is similar to the corrinoid biosynthesis in the anaerobic eubacteria Eubacterium limosum, Clostridium barkeri, and Clostridium thermoaceticum.     

The biosynthesis of the antioxidant caffeic acid derivative subulatin by the liverwort Jungermannia subulata cultured in vitro

The in vitro cultured liverwort Jungermannia subulata produces the unique molecule subulatin. In this study, we examined the incorporation of [1-¹³C] and [1,2-¹³C₂] glucose, [2-¹³C] arabinose, [2-¹³C] caffeic acid, and [1-¹³C] phenylalanine into subulatin. The trilobatinoic acid C unit of subulatin incorporated ¹³C atoms from [1-¹³C] and [1,2-¹³C₂] glucose and from [2-¹³C] arabinose but not from any other of the other precursors. Based on these results and labeling patterns, the trilobatinoic acid C unit of subulatin appears to be biosynthesized from arabinose-5-phosphate and phosphoenolpyruvate.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Biosynthesis of 4-methyl-1-nonanol: Female-produced sex pheromone of the yellow mealworm beetle,Tenebrio molitor (Coleoptera: Tenebrionidae)

The objective of this study was to elucidate the biosynthetic route to 4-methyl-1-nonanol, the female-produced sex pheromone of the yellow mealworm beetle, Tenebrio molitor L. The biosynthetic route to the pheromone was examined by (i) allowing the females to feed on defatted bran coated with a stable isotope-labeled putative precursor ([1-¹³C]acetate, [1-¹³C]propionate, [1-¹³C]pentanoate, [1-¹³C]2-methylheptanoic acid, or [²H₂]4-methylnonanoic acid); (ii) determining if the precursors were incorporated by analyzing the emitted pheromone by gas chromatography/selected ion monitoring-mass spectroscopy (GC/SIM-MS); (iii) where the pheromone was isotopically-enriched, determining the position of the isotopic label(s) through comparison of the MS fragmentation pattern with that of unlabelled 4-methyl-1-nonanol. Although the incorporation of [1-¹³C]acetate into 4-methyl-1-nonanol could not be detected, relatively large proportions of the pheromone were produced from the other precursors tested: 81% from [²H₂]4-methylnonanoic acid, 45% from [1-¹³C]2-methylheptanoic acid, 16% from [1-¹³C]pentanoate, and 35% from [1-¹³C]propionate (27% from only one unit, and 7.8% from two units). The results indicate that 4-methyl-1-nonanol is produced through a modification of normal fatty acid biosynthesis: initiation of the pathway with one unit of propionate results in the uneven number of carbons in the chain; incorporation of another unit of propionate during elongation provides the methyl branch; reduction of 4-methylnonanoic acid produces the alcohol pheromone. The elucidation of the biosynthetic pathway of 4-methyl-1-nonanol biosynthesis in the yellow mealworm is the first step towards understanding the biochemistry of sex pheromone production in this species.     

Two Biosynthetic Pathways to delta-Aminolevulinic Acid in a Pigment Mutant of the Green Alga, Scenedesmus obliquus

Pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus develops only traces of chlorophyll and has no detectable amount of δ-aminolevulinic acid (ALA) when grown in the dark. In light it develops ALA and in the presence of levulinic acid (LA), a competitive inhibitor of ALA dehydratase, it accumulates 0.18 mmoles of ALA per 10 microliters of packed cell volume per 12 hours. This amount could be increased up to 15 times by feeding precursors and cofactors.

Incubation with [U-¹⁴C]glutamate, [1-¹⁴C]glutamate, and [2-¹⁴C]glycine yielded significantly labeled ALA, whereas [1-¹⁴C]glycine did not label the ALA specifically. Thus, two pathways using either glycine/succinyl-coenzyme A or incorporating the whole C-5-skeleton of glutamate into ALA are present in this alga. The efficiency of the glycine/succinyl-coenzyme A pathway seems to be three times higher than that of the glutamate pathway. Incubation with [5-¹⁴C]2-ketoglutarate, which can serve both pathways as a precursor, resulted in radioactivity of ALA as high as the sum of both labeling with [1-¹⁴C]glutamate and [2-¹⁴C]glycine.

Since the newly synthesized chlorophyll was radioactive regardless of labeled substrate employed, both pathways culminate in chlorophyll formation.

     

Design,synthesis, radiolabeling and in vivo evaluation of potential positron emission tomography (PET) radioligands for brain imaging of the 5-HT7 receptor

Here we describe the design, synthesis, and pharmacological evaluation of a set of compounds structurally related to the high affinity serotonin 5-HT₇ receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (6, LP-211). Specific structural modifications were performed in order to maintain affinity for the target receptor and to improve the selectivity over 5-HT_1A and adrenergic α₁ receptors. The synthesized compounds have chemical features that could enable labeling with a positron emitter radioisotope (carbon-11 or fluorine-18) and lipophilicity within the range considered optimal for brain penetration and low non-specific binding. 4-[2-(4-Methoxyphenyl)phenyl]-N-(pyridin-4-ylmethyl)piperazinehexanamide (23a) and N-pyridin-4-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (26a) were radiolabeled on the methoxy group with carbon-11. Positron emission tomography (PET) analysis revealed that [¹¹C]-23a and [¹¹C]-26a were P-glycoprotein (P-gp) substrates and rapidly metabolized, resulting in poor brain uptake. These features were not predicted by in vitro tests.     

Biosynthesis of andrographolide in Andrographis paniculata

Andrographolide, a diterpene lactone, is isolated from Andrographis paniculata which is well known for its medicinal properties. The biosynthetic route to andrographolide was studied using [1-¹³C]acetate, [2-¹³C]acetate and [1,6-¹³C₂]glucose. The peak enrichment of eight carbon atoms in the ¹³C NMR spectra of andrographolide suggested that deoxyxylulose pathway (DXP) is the major biosynthetic pathway to this diterpene.The contribution of the mevalonic acid pathway (MVA) is indicated by the observed ¹³C-labeling pattern, and because the labeling patterns indicate a simultaneous contribution of both methyl erythritol phosphate (MEP) and MVA pathways it can be deduced that cross-talk occurs between plastids and cytoplasm.     

The biosynthesis of the thiazole moiety of thiamine in Salmonella typhimurium

The mechanism of biosynthesis of 4-methyl-5-β-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various ¹⁴C-labeled precursors was determined. We found that [Me-¹⁴C]methionine, [2-¹⁴C]methionine, [U-¹⁴C]alanine, and [2-¹⁴C]glycine were not incorporated whereas [2-¹⁴C]-tyrosine was incorporated. Degradation of the 4-methyl-5-β-hydroxyethyl thiazole obtained after [2-¹⁴C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-β-hydroxyethyl thiazole biosynthesis.     

Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION

The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C₂₀ and C₂₂ fatty acids, which contain an unusual Δ5 double bond. When [1-¹⁴C]acetate was incubated with developing seed slices, ¹⁴C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [¹⁴C]acetate and [¹⁴C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[¹⁴C]11-eicosenoate, cis-13-[¹⁴C]docosenoate, and cis-5,cis-13-[¹⁴C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C₂₀ and C₂₂ acyl species. The Δ5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-¹⁴C-labeled free fatty acids. Using [1-¹⁴C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.     

Chloroplast culture: The chlorophyll repair potential of mature chloroplasts incubated in a simple medium

The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCI/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protochlorophyllide from exogenous δ-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between δ-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from δ-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of δ-amino-[4-¹⁴C]levulinic acid into [¹⁴C] protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E₄₃₀-F₆₃₁)] exhibited a soret excitation maximum at 430 nm (E₄₃₀) and a fluorescence emission maximum at 631 nm (F₆₃₁) in methanol/acetone (4 : 1, v/v). Cp (E₄₃₀-F₆₃₁) was shown to be neither protochlorophyllide nor zinc-protochlorophyllide but an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.     

Real-time Detection of Hepatic Gluconeogenic and Glycogenolytic States Using Hyperpolarized [2-13C]Dihydroxyacetone

Glycogenolysis and gluconeogenesis are sensitive to nutritional state, and the net direction of flux is controlled by multiple enzymatic steps. This delicate balance in the liver is disrupted by a variety of pathological states including cancer and diabetes mellitus. Hyperpolarized carbon-13 magnetic resonance is a new metabolic imaging technique that can probe intermediary metabolism nondestructively. There are currently no methods to rapidly distinguish livers in a gluconeogenic from glycogenolytic state. Here we use the gluconeogenic precursor dihydroxyacetone (DHA) to deliver hyperpolarized carbon-13 to the perfused mouse liver. DHA enters gluconeogenesis at the level of the trioses. Perfusion conditions were designed to establish either a gluconeogenic or a glycogenolytic state. Unexpectedly, we found that [2-¹³C]DHA was metabolized within a few seconds to the common intermediates and end products of both glycolysis and gluconeogenesis under both conditions, including [2,5-¹³C]glucose, [2-¹³C]glycerol 3-phosphate, [2-¹³C]phosphoenolpyruvate (PEP), [2-¹³C]pyruvate, [2-¹³C]alanine, and [2-¹³C]lactate. [2-¹³C]Phosphoenolpyruvate, a key branch point in gluconeogenesis and glycolysis, was monitored in functioning tissue for the first time. Observation of [2-¹³C]PEP was not anticipated as the free energy difference between PEP and pyruvate is large. Pyruvate kinase is the only regulatory step of the common glycolytic-gluconeogenic pathway that appears to exert significant control over the kinetics of any metabolites of DHA. A ratio of glycolytic to gluconeogenic products distinguished the gluconeogenic from glycogenolytic state in these functioning livers.     

Effects of galactose on direct and indirect pathway estimates of hepatic glycogen synthesis

Hepatic glycogen is formed by direct and indirect pathways whose activities reflect altered nutrition or disease. Direct/indirect pathway measurements often involve test meals where ～10% of carbohydrate is galactose, but its effects on direct/indirect pathway estimates are unknown. Therefore, direct/indirect pathway contributions in 24-h fasted rats given 2 g/kg 100% glucose (GLU, n=6) or 90% glucose–10% galactose (GLU+GAL, n=6) were measured by [U-¹³C]glucose dilution and by position-5/position-2 glycogen enrichment (H5/H2) from 2H₂O. For GLU+GAL, galactose glycogenesis was independently measured with [1-¹³C]galactose. Glycogenesis was equivalent in both groups but for GLU+GAL, 23±4% of glycogen was derived from galactose. [U-¹³C]glucose reported a 30±3% direct pathway contribution to glycogenesis for GLU but only 20±3% for GLU+GAL (p=0.012 vs. GLU). H5/H2 yielded identical direct pathway estimates (32±3% GLU, 29±6% GLU+GAL). Thus, galactose glycogenesis was undetected by H5/H2 while [U-¹³C]glucose reported a reduced direct/indirect pathway ratio. With [1-¹³C]galactose also present, correct glycogenic source contributions were obtained.     

Pathway of malic Acid synthesis in response to ion uptake in wheat and lupin roots: evidence from fixation of C and C

Malate synthesis by CO₂ fixation in wheat (Triticum aestivum L.) and lupin (Lupinus luteus) roots was investigated by labeling with NaH¹³CO₃ as well as with NaH¹⁴CO₃. The distribution of ¹⁴C label in the malate was examined, using enzymic degradation methods (malic enzyme, pyruvate decarboxylase) and, in the case of ¹³C, gas chromatography-mass spectrometry. In long-term experiments (2 to 12 hours), both methods showed that the [1-C] and [4-C] positions of malic acid are approximately equally labeled, in agreement with former findings. Short-term experiments (15, 30 seconds) showed that ¹⁴C is confined initially to the [4-C] position of malate but then is distributed quickly to the [1-C] atom. Neither labeling pattern nor rate of randomization was influenced by salt treatment. Analysis of malate from roots by gas chromatography-mass spectrometry, a procedure which was tested against in vitro-prepared [1-¹³C]-, [4-¹³C]-, and [1,4-¹³C] malate, gave strong evidence for the existence of only singly labeled malate molecules. These data suggest that only one carboxylation step, catalyzed by phosphoenolpyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, is responsible for malic acid synthesis in roots and that malate label is randomized by a fumarase-like reaction, presumably in mitochondria.     

Biosynthesis of the iridoid glucoside,lamalbid, in Lamium barbatum

The biosynthesis of the iridoid glucoside lamalbid in Lamium barbatum, a plant species in the Lamiaceae, was investigated by administrating ¹³C-labeled intermediates of MVA and MEP pathways, respectively. The results demonstrated that [3,4,5-¹³C₃]1-deoxy-d-xylulose 5-phosphate could be incorporated into lamalbid, whereas the incorporation of [2-¹³C₁]mevalonolactone was not observed. Based on the ¹³C labeling pattern of lamalbid and the incorporation data, we deduce that the iridoid glucoside in L. barbatum is biosynthesized through the MEP pathway, whereas the classic MVA pathway is not utilized.     

Biosynthesis of podophyllotoxin in Linum album cell cultures

Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding experiments were performed with [2-¹³C]3′,4′-dimethoxycinnamic acid, [2-¹³C]3′,4′-methylenedioxycinnamic acid (MDCA), [2-¹³C]3′,4′,5′-trimethoxycinnamic acid, [2-¹³C]sinapic acid, [2-¹³C]- and [2,3-¹³C₂]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX. These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX. Electronic supplementary material to this article is available at and is accessible for authorized users. Electronic Publication     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

Synthesis and biological evaluation of 11C-labeled β-galactosyl triazoles as potential PET tracers for in vivo LacZ reporter gene imaging

In our aim to develop LacZ reporter probes with a good retention in LacZ expressing cells, we report the synthesis and preliminary evaluation of two carbon-11 labeled β-galactosyl triazoles 1-(β-d-galactopyranosyl)-4-(p-[¹¹C]methoxyphenyl)-1,2,3-triazole ([¹¹C]-6) and 1-(β-d-galactopyranosyl)-4-(6-[¹¹C]methoxynaphthyl)-1,2,3-triazole ([¹¹C]-13). The precursors for the radiolabeling and the non-radioactive analogues (6 and 13) were synthesized using straightforward ‘click’ chemistry. In vitro incubation experiments of 6 with β-galactosidase in the presence of o-nitrophenyl β-d-galactopyranoside (ONPG) showed that the triazolic compound was an inhibitor of β-galactosidase activity. Radiolabeling of both precursors was performed using [¹¹C]methyl iodide as alkylating agent at 70 °C in DMF in the presence of a small amount of base. The log P values were ?0.1 and 1.4, respectively, for [¹¹C]-6 and [¹¹C]-13, the latter therefore being a good candidate for increased cellular uptake via passive diffusion. Biodistribution studies in normal mice showed a good clearance from blood for both tracers. [¹¹C]-6 was mainly cleared via the renal pathway, while the more lipophilic [¹¹C]-13 was excreted almost exclusively via the hepatobiliary system. Despite the lipophilicity of [¹¹C]-13, no brain uptake was observed. Reversed phase HPLC analysis of murine plasma and urine revealed high in vivo stability for both tracers. In vitro evaluation in HEK-293T cells showed an increased cell uptake for the more lipophilic [¹¹C]-13, however, there was no statistically higher uptake in LacZ expressing cells compared to control cells.