Biodistribution and catabolism of 18F-labelled isopeptide Nɛ-(γ-glutamyl)-L-lysine

Summary. Isopeptide bonds between the ɛ-amino group of lysine and the γ-carboxamide group of glutamine are formed during strong heating of pure proteins or, more important, by enzymatic reaction mediated by transglutaminases. Despite the wide use of a microbial transglutaminase in food biotechnology, up to now little is known about the metabolic fate of the isopeptide N^ɛ-(γ-glutamyl)-L-lysine. In the present study, N-succinimidyl-4-[¹⁸F]fluorobenzoate was used to modify N^ɛ-(γ-glutamyl)-L-lysine at each of its two α-amino groups, resulting in the 4-[¹⁸F]fluorobenzoylated derivatives, for which biodistribution, catabolism, and elimination were investigated in male Wistar rats. A significant different biochemical behavior of the two labelled isopeptides was observed in terms of in vitro stability, in vivo metabolism as well as biodistribution. The results suggest that the metabolic fate of isopeptides is likely to be dependent on how they are reabsorbed – free or peptide bound.     

Synthesis and biodistribution of an 18F-labelled resveratrol derivative for small animal positron emission tomography

Summary. Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a naturally occurring phytoalexin and polyphenol existing in grapes and various other plants, and one of the best known ‘nutriceuticals’. It shows a multiplicity of beneficial biological effects, particularly, by attenuating atherogenic, inflammatory, and carcinogenic processes. However, despite convincing evidence from experimental and clinical studies, data concerning the role of resveratrol and other members of the large polyphenols family for human health is still a matter of debate. One reason for this is the lack of suitable sensitive and specific methods, which would allow direct assessment of biodistribution, biokinetics, and the metabolic fate of these compounds in vivo. The unique features of positron emission tomography (PET) as a non-invasive in vivo imaging methodology in combination with suitable PET radiotracers have great promise to assess quantitative information on physiological effects of polyphenols in vivo. Herein we describe the radiosynthesis of an ¹⁸F-labelled resveratrol derivative, 3,5-dihydroxy-4′-[¹⁸F]fluoro-trans-stilbene ([¹⁸F]-1), using the Horner-Wadsworth-Emmons reaction as a novel radiolabelling technique in PET radiochemistry for subsequent functional imaging of polyphenol metabolism in vivo. In a typical “three-step/one-pot” reaction, ¹⁸F-labelled resveratrol derivative [¹⁸F]-1 could be synthesized within 120–130 min including HPLC separation at a specific radioactivity of about 90 GBq/μmol. The radiochemical yield was about 9% (decay-corrected) related to [¹⁸F]fluoride and the radiochemical purity exceeded 97%. First radiopharmacological evaluation included measurement of biodistribution ex vivo and positron emission tomography (PET) studies in vivo after intravenous application of [¹⁸F]-1 in male Wistar rats using a dedicated small animal PET camera with very high spatial resolution. Concordantly with data on bioavailability and metabolism of native resveratrol from the literature, these investigations revealed an extensive uptake and metabolism in the liver and kidney, respectively, of [¹⁸F]-1. This study represents the first investigation of polyphenols in vivo by means of PET.     

Systematic comparison of two novel,thiol-reactive prosthetic groups for <Superscript>18</Superscript>F labeling of peptides and proteins with the acylation agent succinimidyl-4-[<Superscript>18</Superscript>F]fluorobenzoate ([<Superscript>18</Superscript>F]SFB)

A systematic comparison of 4-[¹⁸F]fluorobenzaldehyde-O-(2-{2-[2-(pyrrol-2,5-dione-1-yl)ethoxy]-ethoxy}-ethyl)oxime ([¹⁸F]FBOM) and 4-[¹⁸F]fluorobenzaldehyde-O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]oxime ([¹⁸F]FBAM) as prosthetic groups for the mild and efficient ¹⁸F labeling of cysteine-containing peptides and proteins with the amine-group reactive acylation agent, succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB), is described. All three prosthetic groups were prepared in a remotely controlled synthesis module. Synthesis of [¹⁸F]FBOM and [¹⁸F]FBAM was accomplished via oxime formation through reaction of appropriate aminooxy-functionalized labeling precursors with 4-[¹⁸F]fluorobenzaldehyde. The obtained radiochemical yields were 19% ([¹⁸F]FBOM) and 29% ([¹⁸F]FBAM), respectively. Radiolabeling involving [¹⁸F]FBAM and [¹⁸F]FBOM was exemplified by the reaction with cysteine-containing tripeptide glutathione (GSH), a cysteine-containing dimeric neurotensin derivative, and human native low-density lipoprotein (nLDL) as model compounds. Radiolabeling with the acylation agent [¹⁸F]SFB was carried out using a dimeric neurotensin derivative and nLDL. Both thiol-group reactive prosthetic groups show significantly better labeling efficiencies for the peptides in comparison with the acylation agent [¹⁸F]SFB. The obtained results demonstrate that [¹⁸F]FBOM is especially suited for the labeling of hydrophilic cysteine-containing peptides, whereas [¹⁸F]FBAM shows superior labeling performance for higher molecular weight compounds as exemplified for nLDL apolipoprotein constituents. However, the acylation agent [¹⁸F]SFB is the preferred prosthetic group for labeling nLDL under physiological conditions.     

Aspects of positron emission tomography radiochemistry as relevant for food chemistry

Summary. Positron emission tomography (PET) is a medical imaging technique using compounds labelled with short-lived positron emitting radioisotopes to obtain functional information of physiological, biochemical and pharmacological processes in vivo. The need to understand the potential link between the ingestion of individual dietary agents and the effect of health promotion or health risk requires the exact metabolic characterization of food ingredients in vivo. This exciting but rather new research field of PET would provide new insights and perspectives on food chemistry by assessing quantitative information on pharmocokinetics and pharmacodynamics of food ingredients and dietary agents. To fully exploit PET technology in food chemistry appropriately radiolabelled compounds as relevant for food sciences are needed. The most widely used short-lived positron emitters are ¹¹C (t_1/2 = 20.4 min) and ¹⁸F (t_1/2 = 109.8 min). Longer-lived radioisotopes are available by using ⁷⁶Br (t_1/2 = 16.2 h) and ¹²⁴I (t_1/2 = 4.12 d). The present review article tries to discuss some aspects for the radiolabelling of food ingredients and dietary agents either by means of isotopic labelling with ¹¹C or via prosthetic group labelling approaches using the positron emitting halogens ¹⁸F, ⁷⁶Br and ¹²⁴I.     

Site-selective radiolabeling of peptides by 18F-fluorobenzoylation with [18F]SFB in solution and on solid phase: a comparative study

Peptides labeled with short-lived positron-emitting radionuclides are of outstanding interest as probes for molecular imaging by positron emission tomography (PET). Herein, the site-selective incorporation of fluorine-18 into lysine-containing peptides using the prosthetic labeling agent N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) is described. The reaction of [¹⁸F]SFB with four biologically relevant resin-bound peptides was studied and optimized. For comparison, each peptide was ¹⁸F-fluorobenzoylated in solution under different conditions and the product distribution was analyzed confirming the advantages of the solid-phase approach. The method’s feasibility for selective radiolabeling either at the N-terminus or at the lysine side chain was demonstrated. Labeling on solid phase with [¹⁸F]SFB resulted in crude ¹⁸F-fluorobenzoylpeptides whose radiochemical purities were typically greater than 90% and that could be prepared in synthesis times from 65 to 76?min.     

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Biomolecules, including peptides,^1-9 proteins,^10,11 and antibodies and their engineered fragments,^12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.^15-18 Therefore, significant effort has devoted to the synthesis and exploration of ¹⁸F-labeled biomolecules. Although there are elegant examples of the direct ¹⁸F-labeling of peptides,^19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.^23,24N-Succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB),^25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the ¹⁸F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [¹⁸F]SFB, the resulting [¹⁸F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.¹ Most [¹⁸F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [¹⁸F]SFB syntheses have been reported,^{29-32, 41-42} they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [¹⁸F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.Herein, we report a concise preparation of [¹⁸F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,^38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.³⁸ Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.^28,43 The novelty of our improved [¹⁸F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [¹⁸F]SFB.Download video file.^{(51M, mov)}     

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells

Summary. The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK₁ cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [¹⁴C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [¹⁴C] L-DOPA handling. The basal-to-apical transepithelial flux of [¹⁴C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [¹⁴C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [¹⁴C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [¹⁴C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [¹⁴C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [¹⁴C] L-DOPA through the apical or the basal cell side. It is likely that the Na⁺-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.     

Metabolic fates of 2′-deoxy-5-[18F]fluorouridine in tumor-bearing mice and human plasma

The metabolic fate of 2′-deoxy-5-[¹⁸F]fluorouridine ([¹⁸F]FdUrd), a useful positron emission tomography (PET) tracer of nucleic acid metabolism in tumors, was investigated in mice and humans. A rapid increase in labeled catabolites was found in mouse and human plasma. In mouse FM3A mammary carcinoma, the corresponding catabolites were also detected in addition to metabolites which were activated by the nucleic acid metabolism. From a biodistribution study of β-[³H]alanine, α-[¹⁸F]fluoro-β-alanine, a major catabolite, was assumed to be taken up twice as much by tumor than by the brain. Nucleic acid metabolism in brain tumors by [¹⁸F]FdUrd-PET may be assessed using normal brain regions as a reference.     

Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS+nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS+nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions (()O(2)(-)) and peroxynitrite (ONOO(-)) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS+nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS+nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation.     

Modulation of taurine uptake in the goldfish retina and axonal transport to the tectum¶Effect of crushing the optic nerve or axotomy

Summary. Although there are a great number of studies concerning the uptake of taurine in several tissues, the regulation of taurine transport has not been studied in the retina after lesioning the optic nerve. In the present study, isolated retinal cells of the goldfish retina were used either immediatly after cell suspension or in culture. The high-affinity transport system of [³H]taurine in these cells was sodium-, temperature- and energy-dependent, and was inhibited by hypotaurine and β-alanine, but not by γ-aminobutyric acid. There was a decrease in the maximal velocity (V_max) without modifications in the substrate affinity (K_m) after optic axotomy. These changes were mantained for up to 15 days after the lesion. The results might be the summation of mechanisms for providing extracellular taurine to be taken up by other retinal cells or eye structures, or regulation by the substrate taurine, which increases after lesioning the optic nerve. The in vivo accumulation of [³H]taurine in the retina after intraocular injection of [³H]taurine was affected by crushing the optic nerve or by axotomy. A progressive retinal decrease in taurine transport was observed after crushing the optic nerve, starting at 7 hours after surgery on the nerve. The uptake of [³H]taurine by the tectum was compensated in the animals that were subjected to crushing of the optic nerve, since the concentration of [³H]taurine was only different from the control value 24 hours after the lesion, indicating an efficient transport by the remaining axons. On the contrary, the low levels of [³H]taurine in the tectum after axotomy might be an index of the non-axonal origin of taurine in the tectum. Axonal transport was illustrated by the differential presence of [³H]taurine in the intact or crushed optic nerve. The uptake of [³H]taurine into retinal cells in culture in the absence or in the presence of taurine might indicate the existence of an adaptive regulation of taurine transport in this tissue, however taurine transport probably differentially occurs in specific populations of retinal cells. The use of a purified preparation of cells might be useful for future studies on the modulation of taurine transport by taurine in the retina and its role during regeneration. Received June 11, 1999/Accepted August 31, 1999     

In vitro biosynthesis of 1,4-β-galactan attached to rhamnogalacturonan I

 The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[¹⁴C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [¹⁴C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-β-galactanase released 65–70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [¹⁴C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-β-galactanase. Thus, the majority of the ¹⁴C-labelled product was 1,4-β-galactan. Compounds released by the endo-1,4-β-galactanase treatment were mainly [¹⁴C]galactose and [¹⁴C]galactobiose, indicating that the synthesized 1,4-β-galactan was longer than a trimer. In vitro synthesis of 1,4-β-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn²⁺. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [¹⁴C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-β-galactanase, radioactivity was not only found as [¹⁴C]galactose or [¹⁴C]galactobiose but also as larger fragments. The larger fragments were likely the [¹⁴C]galactose or [¹⁴C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with β-galactosidase together with endo-1,4-β-galactanase digested all radioactivity to the fraction eluting as [¹⁴C]galactose. The data indicate that the majority of the [¹⁴C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-β-galactan sidechains in the endogenous pectic rhamnogalacturonan I. Received: 24 June 1999 / Accepted: 30 August 1999     

Expression,purification and fluorine-18 radiolabeling of recombinant S100A4: a potential probe for molecular imaging of receptor for advanced glycation endproducts in vivo?

Data concerning the pathophysiological role of extracellular S100A4, a member of the multigenic family of Ca²⁺-modulated S100 proteins, and its interaction with the receptor for advanced glycation endproducts (RAGE) or other putative receptors in tumorigenesis, metastasis, and inflammatory processes in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A4 as potential probe for molecular imaging and functional characterization of this interaction. Therefore, human S100A4 was cloned as GST fusion protein in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100A4 was radiolabeled with the positron emitter fluorine-18 (¹⁸F) by conjugation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The radioligand [¹⁸F]fluorobenzoyl-S100A4 (¹⁸F-S100A4) was used in cell binding experiments in RAGE-bearing human melanoma cells and endothelial cells in vitro, and in both biodistribution experiments and small animal positron emission tomography (PET) studies in normal rats in vivo. The cellular association and tissue-specific distribution of ¹⁸F-S100A4 in vitro and in vivo correlated well with the protein expression and anatomical localization of RAGE, e.g., in the vascular system and in lung. Compared to other S100 RAGE radioligands, the overall findings of this study indicate that extracellular S100A4 in vivo shows only a moderate interaction with RAGE and, furthermore, exhibits a substantially faster metabolic degradation. On the other hand, the approach allows the use of quantitative small animal PET and provides a novel probe to both delineate functional expression and differentiate multiligand interaction of RAGE under normal and pathophysiological conditions in rodent models of disease.     

Low-density lipoproteins induce the renin-angiotensin system and their receptors in human endothelial cells.

Increased levels of low-density lipoproteins are well-established risk factors of endothelial dysfunction and the metabolic syndrome. In this study, we evaluated the effect of native low-density lipoprotein (nLDL) and oxidized LDL (oxLDL) on the expression of genes of the renin-angiotensin system (angiotensin-converting enzyme, ACE; angiotensin II type 1 receptor, AT(1)) and their receptors (low-density lipoprotein receptor: LDLR; lectin-like oxLDL receptor: LOX-1; toll-like receptor 4: TLR4) in primary cultures of human umbilical vein endothelial cells. ACE and AT(1) expressions were significantly increased after stimulation with nLDL and oxLDL. OxLDL receptor LOX-1 showed a maximum induction after 7 hours. Increased LOX-1 protein expression in response to oxLDL could be blocked by a LOX-1-specific antibody. TLR4 expression was increased by nLDL and oxLDL as well. We conclude that LDL and oxLDL can activate the renin-angiotensin system and their receptors LDLR, LOX-1, and TLR4 in human endothelial cells. These data suggest a novel link between hypercholesterolemia and hypertension in patients with the metabolic syndrome.     

Effect of γ-radiation on the ratio of [18F]2-fluoro-2-deoxy-D-glucose to glucose utilization in human glioblastoma cellsin vitro

The glucose consumption in tumoursin vivo as reflected by uptake of [¹⁸F]2-fluoro-2-deoxy-D-glucose (¹⁸FDG) using positron emission tomography (PET) is currently under investigation as a measure of tumour response to radiotherapy. The calculation of cerebral metabolic rate of glucose from¹⁸FDG-PET data requires a proportionality factor referred to as the lumped constant. In the presentin vitro study, the utilizations of¹⁸FDG and glucose have been measured in a human glioblastoma cell line (86HG-39) as a function of γ-radiation dose with various post-irradiation times and of different fractionation modes. The ratio of utilization of¹⁸FDG to that of glucose (R_F/G), assumed to correspond to the lumped constant, was observed to increase 12 and 24 h after single fraction γ-exposure by factors ranging from 1.2 to 1.5 compared with the non-irradiated controls. It decreased after multiple fraction γ-exposure (4 × 2 Gy) by a factor of 0.7 compared with the single fraction schedule (1 × 8 Gy). The results suggest that the affinities of glucose transporters or hexokiriase enzyme or both for¹⁸FDG and glucose could be influenced by γ-irradiation in this tumour cell linein vitro. Apparent changes of the glucose consumption determined with PET in human tumours following radiotherapy may, therefore, not be solely due to changes in cellular metabolism or cell number but may also be due to changes in R _F/G .     

Tumor uptake study of 18F-labeled N-acetylneuraminic acids

In order to develop positron-emitting tracers for imaging metabolic functions of tumors with positron emission tomography, tumor uptake of N-acetyl-3-[¹⁸F]fluoroneuraminic acid and N-acetyl-2-deoxy-2,3-di-[¹⁸F fluoroneuraminic acid was investigated in mice or rats. The two tracers showed similar tissue distribution patterns. After i.v. injection of each tracer into mice with an FM3A tumor, the radioactivity was very rapidly cleared from normal and tumor tissues. Only tumor-to-brain and tumor-to-muscle uptake ratios were greater than 1.0 for 2 h. In 7 types of tumor models, no selective tumor uptake of tracers was observed 30 min after injection. The metabolic alteration rate of N-acetyl-3-[¹⁸F]fluoroneuraminic acid in FM3A, liver and kidney was very slow. Neither tracer may be suitable for tumor imaging in vivo.     

Accumulation of 2-deoxy-2-[18F]fluoro-d-galactose in the liver by phosphate and uridylate trapping

To investigate the highest accumulation of 2-deoxy-2-[¹⁸F]fluoro-d-galactose ([¹⁸F]FdGal) in the liver, metabolic studies with [¹⁸F]FdGal were carried out in Wistar rats for 120 min after i.v. injection. As main metabolites 2-deoxy-2-[¹⁸F]fluoro-d-galactose 1-phosphate ([¹⁸F]FdGal-1-P) and UDP-2-deoxy-2-[¹⁸F]-fluoro-d-galactose (UDP-[¹⁸F]FdGal) were identified in the liver and other tissues. The [¹⁸F]FdGal was phosphorylated by galactokinase. The phosphorylation rate was very rapid in the liver, in which at 5 min after injection 81% of ¹⁸F was detected as [¹⁸F]FdGal-1-P. After this time the phosphate form decreased with time, which was explained by conversion of [¹⁸F]FdGal-1-P to UDP-[¹⁸F]FdGal by UDP-glucose: galactose-1-phosphate uridyltransferase. At 120 min after injection 77% of the ¹⁸F was measured in the UDP-[¹⁸F]FdGal. In the brain both reaction rates were slower than in the liver. Both phosphate and uridylate derivates were also observed as main metabolites in the heart, lung, spleen and small intestine. On the other hand, a small amount of [¹⁸F]FdGal-1-P was detected in the plasma, in which the percentage of phosphate increased gradually and was 6% at 120 min.These results show that the [¹⁸F]FdGal metabolism in tissue results in phosphate and uridylate trapping and that the [¹⁸F]FdGal has potential for measuring in vivo galactose metabolism with positron emission tomography.     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

PET imaging of liposomes labeled with an [18F]-fluorocholesteryl ether probe prepared by automated radiosynthesis

A novel [¹⁸F]-labeled cholesteryl ether lipid probe was prepared by synthesis of the corresponding mesylate, which was [¹⁸F]-fluorinated by a [¹⁸F]KF, Kryptofix-222, K₂CO₃ procedure. Fluorination was done for 10 minutes at 165^°C and took place with conversion between 3 and 17%, depending on conditions. Radiolabelling of the probe and subsequent in situ purification on SEP-Paks were done on a custom-built, fully automatic synthesis robot. Long-circulating liposomes were prepared by hydration (magnetic stirring) of a lipid film containing the radiolabeled probe, followed by fully automated extrusion through 100-nm filters. The [¹⁸F]-labeled liposomes were injected into nude, tumor-bearing mice, and positron emission tomography (PET) scans were performed several times over 8 hours to investigate the in vivo biodistribution. Clear tumor accumulation, as well as hepatic and splenic uptake, was observed, corresponding to expected liposomal pharmacokinetics. The tumor accumulation 8 hours postinjection accounted for 2.25?±?0.23 (mean ± standard error of the mean) percent of injected dose per gram (%ID/g), and the tumor-to-muscle ratio reached 2.20?±?0.24 after 8 hours, which is satisfactorily high for visualization of pathological lesions. Moreover, the blood concentration was still at a high level (13.9?±?1.5 %ID/g) at the end of the 8-hour time frame. The present work demonstrates the methodology for automated preparation of radiolabeled liposomes, and shows that [¹⁸F]-labeled liposomes could be suitable as a methodology for visualization of tumors and obtaining short-term pharmacokinetics in vivo.     

Principles of quantitative positron emission tomography

Summary. The central distinguishing feature of positron emission tomography (PET) is its ability to investigate quantitatively regional cellular and molecular transport processes in vivo with good spatial resolution. This review wants to provide a concise overview of the established principles underlying quantitative data evaluations of the acquired PET images. Especially, the compartment modelling framework is discussed on which virtually all quantification methods utilized in PET are based. The aim of the review is twofold: first, to provide the reader with an idea of the theoretical framework and mathematical tools and second, to enable an intuitive grasp of the possibilities and limitations of a quantitative approach to PET data evaluation. This should facilitate an understanding of how PET measurements translate into quantities such as regional blood flow, volume of distribution, and metabolic rates of specific substrates.     

Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose derivative [18F]FPGal for imaging the hepatic asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it recognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic distribution, the asialoglycoprotein receptor can be targeted by positron imaging agents to study liver function using PET imaging. In this study, the positron imaging agent [¹⁸F]FPGal was designed to specifically target hepatic asialoglycoprotein receptor and its effectiveness was assessed in in vitro and in vivo models. The radiosynthesis of [¹⁸F]FPGal required 50 min with total radiochemical yields of [¹⁸F]FPGal from [¹⁸F]fluoride as 10% (corrected radiochemical yield). The K_d of [¹⁸F]FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM. Uptake values of 0.55% were observed within 30 min of incubation with HepG2 cells, which could be blocked by 200 mM d(+)-galactose (<0.1%). In vivo biodistribution analysis showed that the liver accumulation of [¹⁸F]FPGal at 30 min was 4.47 ± 0.96% ID/g in normal mice compared to 1.33 ± 0.07% ID/g in hepatic fibrotic mice (P < 0.01). Reduced uptake in the hepatic fibrosis mouse models was confirmed through PET/CT images at 30 min. Compared to normal mice, the standard uptake value (SUV) in the hepatic fibrosis mice was significantly lower when assessed through dynamic data collection for 1 h. Therefore, [¹⁸F]FPGal is a feasible PET probe that provide insight into ASGPR related liver disease.