Pattern Of Radioactivity Research Articles

Preferential uptake of the non steroid anti-inflammatory drug diclofenac into inflamed tissues after a single oral dose in rats

BackgroundDiclofenac is a nonsteroidal anti-inflammatory drug which is available as prescription (RX) and over-the-counter (OTC) medication for the systemic and topical treatment of painful and inflammatory conditions such as arthritis and back pain. This study was undertaken to investigate the distribution and retention of diclofenac and/or its metabolites in inflamed tissues, using the carrageenan-induced inflammation model and quantitative whole body autoradiography in rats.Methods[14C]diclofenac sodium was administrated as a single 2 mg/kg oral dose 1 h after injection of carrageenan into one front and one hind footpads and subcutaneously into the dorsum of the neck of rats. A control animal received saline injections. Three carrageenan-treated rats and one control rat were sacrificed at 1, 4, 8, and 24 h after [14C]diclofenac sodium administration (total of 4 rats/time point). The carcasses were immediately snap-frozen and prepared for cryosectioning. Lengthwise whole-body sections (40 μm thick), including all major tissues, were obtained from different levels across the body. The tissue concentrations of total radiolabeled components were determined using quantitative autoradioluminography.ResultsThe radioactivity patterns demonstrated that diclofenac and/or its metabolites preferentially distributed into the inflamed tissues. In unharmed tissues the distribution was similar in control and treated animals. The exposure, based on the areas under the tissue concentration versus time (AUC0-tlast), was 26 and 53 fold higher in the inflamed neck and inflamed footpads of treated animals than in control rats; the exposures in unharmed tissues were similar in the treated and control rats, and the AUC0-tlast was 17 fold higher in the inflamed paws than in the non inflamed footpads of the carrageenan-treated rats. The higher exposure in the inflamed tissues may be explained partly to the fact that the elimination of total radiolabeled components from inflamed tissues (t1/2 = 6 h) appeared lower than from the corresponding unharmed tissues (t1/2 = 2 h).ConclusionThis animal study demonstrated that diclofenac and/or its metabolites were rapidly and preferentially taken up and retained in inflamed tissues. Although there were theoretical considerations that mildly acidic NSAID may show some preferential distribution in inflamed tissues there was no clear experimental proof for diclofenac until the present study.

Renal uptake and retention of radiolabeled somatostatin, bombesin, neurotensin, minigastrin and CCK analogues: species and gender differences

During therapy with radiolabeled peptides, the kidney is most often the critical organ. Newly developed peptides are evaluated preclinically in different animal models before their application in humans. In this study, the renal retention of several radiolabeled peptides was compared in male and female rats and mice. After intravenous injection of radiolabeled peptides [somatostatin, cholecystokinin (CCK), minigastrin, bombesin and neurotensin analogues], renal uptake was determined in both male and female Lewis rats and C57Bl mice. In addition, ex vivo autoradiography of renal sections was performed to localize accumulated radioactivity. An equal distribution pattern of renal radioactivity was found for all peptides: high accumulation in the cortex, lower accumulation in the outer medulla and no radioactivity in the inner medulla of the kidneys. In both male rats and mice, an increasing renal uptake was found: [(111)In-DTPA]CCK8<[(111)In-DTPA-Pro(1),Tyr(4)]bombesin approximately [(111)In-DTPA]neurotensin<[(111)In-DTPA]octreotide<<[(111)In-DTPA]MG0. Renal uptake of [(111)In-DTPA]octreotide in rats showed no gender difference, and renal radioactivity was about constant over time. In mice, however, renal uptake in females was significantly higher than that in males and decreased rapidly over time in both genders. Moreover, renal radioactivity in female mice injected with [(111)In-DTPA]octreotide showed a different localization pattern. Regarding the renal uptake of different radiolabeled peptides, both species showed the same ranking order. Similar to findings in patients, rats showed comparable and constant renal retention of radioactivity in both genders, in contrast to mice. Therefore, rats appear to be the more favorable species for the study of the renal retention of radioactivity.

Comparison of different amines for the one-lane sequence determination of 5′-end-labeled oligodeoxyribonucleotides by chemical cleavage

Hot aqueous solutions of a wide variety of aliphatic amines bring about efficient cleavage of 5′- 32P-labeled oligodeoxyribonucleotides. Electrophoretic resolution of the product mixtures produces a radioactivity pattern that can be analyzed in terms of relative band intensities and band separations to deduce the base sequence of the original oligonucleotide. Amines differ in their overall reactivity with respect to the oligonucleotide as well as in their preference for reaction with the various heterocyclic bases. Guanine sites, in particular, vary markedly in their cleavage susceptibility when reacted with the different amines, being more vulnerable to scission when reacted with less basic amines. Guanine cleavage propensity can also be affected markedly by the ionic strength of the aminolysis solution. Both effects are interpreted in terms of variable extents of deprotonation of the guanine sites in the basic medium. Some of the amines produce fragment patterns that are marred by the presence of minor extraneous bands; these probably are due to an incomplete extent of the second β-elimination involved in backbone cleavage. The methodology is applicable to confirmatory sequencing of synthetic oligonucleotides and can also be used to prepare standard ladders for use in footprinting experiments or chemical reactivity studies.

Ceramide and sphingomyelin species of fibroblasts and neurons in culture

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.

Diaphragmatic Paralysis Simulating Pulmonary Infection on Gallium-67 Scan

Ga-67 is a useful agent for imaging infection, especially in the chest, but is not so reliable in the abdomen because of physiological bowel activity. A 64-year-old man with fever of unknown origin received a Ga-67 scan for survey of any occult infection focus. Focal Ga-67 accumulation was noted in the left lower chest region on the 24-hour image, but a different pattern of radioactivity was shown on the 48-hour image after bowel cleansing. On review of the chest radiographs and history, we found bowel activity in the left lower chest secondary to diaphragmatic paralysis masqueraded as pulmonary infection. Diaphragmatic paralysis should be considered as a differential diagnosis of abnormal activity in the chest on Ga-67 scan.

The distribution pattern of radioactivity across different tissues in quantitative whole-body autoradiography (QWBA) studies

This study explored whether common rules exist for the distribution patterns across tissues in tissue distribution studies. To investigate this we tested whether tissue:plasma partition coefficients (PCs) of radioactivity are correlated with muscle:plasma PCs. The relationships between PCs of radioactivity in muscle and those in other tissues were investigated in 25 tissues for 20 structurally unrelated drug candidates. Tissue distribution data were obtained by quantitative whole-body autoradiography. Linear regression analysis was performed for each tissue. Radioactivity from basic and acidic/neutral compounds was analyzed separately. Results for acidic/neutral compounds: for the majority of the tissues investigated, the tissue:plasma PCs were well correlated with muscle:plasma PCs ( R 2 > 0.7). Correlations were worse ( R 2 < 0.7) in blood, white fat, excretory organs and tissues protected by a penetration barrier (e.g. brain). Slope factors for the regression ranged from 0.2 (blood) to 3.8 (Harderian gland) and were correlated with neutral lipid contents in tissues. Results for basic compounds: in most tissues, slope factors appeared to be higher than for acidic/neutral compounds. Correlations, however, were poorer than for acidic/neutral compounds. Overall, the present study demonstrates that muscle:plasma PCs are indicative of the overall tissue distribution of drug-related material, as they are well correlated with tissue:plasma PCs in most other tissues. Correlations for acidic/neutral compounds differ from those for basic compounds. The found PC relationships provide an explanation for the distribution pattern across tissues usually seen in tissue distribution studies.

Studies on Blood-Groups AEnzymes.1 and A2

Blood-group O and A2 erythrocytes were treated with the A1-gene-dependent and A2-gene-dependent N-acetylgalactosaminyl transferases in the present of UDP-N-acetyl[14C]galactosamine. Although the transfer of N-acetyl[14C]galactosamine with A2 transferase was slower than with A1 enzyme, group O as well as A2 cells became agglutinable by anti-A1 reagents when incubated with both transferases. Fractionation of the labelled erythrocyte stroma into glycoprotein and glycolipid components showed an approximately equal distribution pattern of radioactivity in all experiments. Likewise, when the short-chain glycolipids and polyglycosylceramides isolated from the labelled stroma were further analyzed by thin-layer chromatography, no major differences were detected in the chromatographic profiles of O and A2 cells when treated with either transferase. These observations indicate that (a) the blood-group-H-type oligosaccharide chains of A2 cells may be similar to those of group-O cells and (b) the serological differences between A1 and A2 cells are likely to be due to a lower density of A-antigenic sites on A2 cells.

Radioactivity in the Ogallala and Dockum Aquifers, northwest Texas, USA

This study investigated distributions of alpha and beta activity in the Ogallala and Dockum Aquifers of northwest Texas, USA. The survey included 319 samples from the Ogallala Aquifer and 48 samples from the Dockum Aquifer. The Ogallala Aquifer produced slightly higher median alpha (7.2 versus 6.1 pCi/L) and beta (7.7 versus 7.0 pCi/L) levels. However, a higher percentage of samples from the Dockum Aquifer exceeded drinking water standards, and the maximum alpha observation from that aquifer was 197 pCi/L, more than 13 times the 15-pCi/L standard. Approximately 4.4% of alpha observations from the Ogallala Aquifer and 16.7% of observations from the Dockum Aquifer exceeded the standard. Fewer observations, 0% from the Ogallala Aquifer and 6.3% from the Dockum Aquifer, exceeded the 50-pCi/L level of concern for beta activity. There were significant positive correlations between alpha and beta activity in each aquifer. However, there were no significant correlations between radioactivity and well depth, other than a weak inverse correlation between beta activity and well depth in the Ogallala Aquifer. Uranium deposits in sandstone and shale of the Dockum Aquifer, and in calcrete and silcrete of the Ogallala Aquifer, and overlying lacustrine sediments, likely influence radioactivity patterns observed in this study.

Disposition and pharmacokinetics of temozolomide in rat

1. Temozolomide, an imidazotetrazine derivative, is a cytotoxic alkylating agent of broad-spectrum antitumour activity. The absorption, metabolism, distribution and excretion of temozolomide have been investigated in male and female Sprague–Dawley and Long–Evans rats following single oral or intravenous dose administration of 200 mg m−2 non-radiolabelled or 14C-radiolabelled temozolomide. The distribution of 14C-temozolomide was also evaluated by whole-body autoradiography in male Sprague–Dawley rats. Plasma concentrations of temozolomide and its active metabolite 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) were determined by high-performance liquid chromatography (HPLC) with ultraviolet detection. Plasma, urine and faeces were profiled by HPLC with radiochemical detection.2. Temozolomide was rapidly and extensively (>90%) absorbed and widely distributed in tissues. The distribution pattern of radioactivity was gender independent. Penetration into the brain following oral or intravenous administration was 35–39% based on the brain/plasma AUC ratio.3. Following intravenous or oral administration, temozolomide was primarily eliminated renally (75–85% of the dose) as either unchanged drug, a carboxylic acid analogue, AIC (a degradation product) and a highly polar unidentified peak. Biliary excretion was minimal (1.4–1.6%). The pharmacokinetics (oral versus intravenous) were similar and gender independent. The absolute oral availability was 96–100%. Temozolomide was rapidly eliminated (t1/2 = 1.2 h) and converted to MTIC.4. Systemic exposure to MTIC was about 2% that of temozolomide. Overall, the disposition of temozolomide in rats was similar to that observed in humans.

An investigation of dopaminergic metabolites in the striatum and in the substantia nigra in vivo utilising radiolabelled L-DOPA and high performance liquid chromatography: a new approach in the search for transmitter metabolites

Although the major routes of dopamine metabolism seem to be established, at least in terminal regions such as the striatum, it is important to search for previously unknown metabolites and to investigate the relevance of previously suggested minor alternative pathways. An urgent issue is to verify and quantify the transformation of dopamine to putative toxic species, another is to further explore metabolism of dopamine located in cell bodies/dendrites, e.g. in the substantia nigra. We have developed a new method in order to widen the search for alternative metabolites of dopamine. The method is based on systemic injection of tritiated L-DOPA to rats in vivo. Brain tissue was homogenised and centrifuged and the resulting supernatant fractioned following passage through a liquid chromatography system. The radioactivity of each fraction was measured using a scintillation system. By identifying fractions containing major catecholamines and metabolites, according to a standard solution, novel metabolites can be searched for in the remaining fractions. It was possible to obtain sufficient radioactivity in separate fractions of supernatant of homogenised tissue, even from such a small brain nucleus as substantia nigra. Radioactivity was obtained in those fractions that contained the major catecholamines and their metabolites, as well as in other fractions where it may represent previously unknown metabolites of L-DOPA/dopamine. The method was used to evaluate the possibility that cytochrome P450 2E1 is involved in the metabolism of dopamine in the substantia nigra. Significant changes in the radioactivity pattern were induced by inhibition of the enzyme but conclusions about whether cytochrome P450 2E1 is involved in the metabolism of dopamine or not requires further studies. The method can be used to study the metabolism of dopamine and can be extended, by using other radiolabelled precursors, also to evaluate metabolism of other transmitters, e.g. serotonin.

N-[ 18F]fluoroethyl-4-piperidyl acetate ([ 18F]FEtP4A) : A PET tracer for imaging brain acetylcholinesterase in vivo

N-[ 18F]Fluoroethyl-4-piperidyl acetate ([ 18F]FEtP4A) was synthesized and evaluated as a PET tracer for imaging brain acetylcholinesterase (AchE) in vivo. [ 18F]FEtP4A was previously prepared by reacting 4-piperidyl acetate (P4A) with 2-[ 18F]fluoroethyl bromide ([ 18F]FEtBr) at 130 °C for 30 min in 37% radiochemical yield using an automated synthetic system. In this work, [ 18F]FEtP4A was synthesized by reacting P4A with 2-[ 18F]fluoroethyl iodide ([ 18F]FEtI) or 2-[ 18F]fluoroethyl triflate ([ 18F]FEtOTf in improved radiochemical yields, compared with [ 18F]FEtBr under the corresponding condition. Ex vivo autoradiogram of rat brain and PET summation image of monkey brain after iv injection of [ 18F]FEtP4A displayed a high radioactivity in the striatum, a region with the highest AchE activity in the brain. Moreover, the distribution pattern of 18F radioactivity was consistent with that of AchE in the brain: striatum>frontal cortex>cerebellum. In the rat and monkey plasma, two radioactive metabolites were detected. However, their presence might not preclude the imaging studies for AchE in the brain, because they were too hydrophilic to pass the blood–brain barrier and to enter the brain. In the rat brain, only [ 18F]fluoroethyl-4-piperidinol ([ 18F]FEtP4OH) was detected at 30 min postinjection. The hydrolytic [ 18F]FEtP4OH displayed a slow washout and a long retention in the monkey brain until the PET experiment (120 min). Although [ 18F]FEtP4A is a potential PET tracer for imaging AchE in vivo, its lower hydrolytic rate and lower specificity for AchE than those of [ 11C]MP4A may limit its usefulness for the quantitative measurement for AchE in the primate brain.

Distribution of adenosine A 2A receptor antagonist KW-6002 and its effect on gene expression in the rat brain

A novel adenosine A 2A receptor selective antagonist, KW-6002 [( E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione], possesses antiparkinsonian activities in rodent and primate models. In the present study, we investigated the distribution of [ 14C]KW-6002 in forebrain after oral administration at pharmacologically effective doses. Also, we monitored the effects of the compound on preproenkephalin (PPE) and preprotachykinin (PPT) gene expression in rat striatum. The highest level of radioactivity was observed in the striatum after oral administration of [ 14C]KW-6002; 30 min after 0.1 and 0.3 mg/kg, the density values in the striatum were 2.45 and 2.43 times higher than those in a reference region (frontal cortex), respectively. At the dose of 3 mg/kg, p.o., the ratio was only 1.58 and the compound was distributed more extensively in the brain. The distribution pattern and intensity of radioactivity were maintained even 90 min after the administration of [ 14C]KW-6002. Oral administration of KW-6002 (0.3 and 3 mg/kg/day) to rats for 14 days reversed the increased gene expression of PPE in striatum that had been depleted of dopamine by prior treatment with 6-hydroxydopamine (6-OHDA). On the other hand, KW-6002 did not alter the decreased gene expression of PPT in 6-OHDA-treated rats. These results are the first to show directly that orally administered KW-6002 is distributed selectively to the striatum and that it modulates the activity of striatopallidal enkephalin-containing neurons but not striatonigral substance P-containing neurons.

Synthesis and evaluation of 3-(4-chlorobenzyl)-8-[ 11C]methoxy-1,2,3,4-tetrahydrochromeno[3,4- c]pyridin-5-one: a PET tracer for imaging sigma 1 receptors

3-(4-Chlorobenzyl)-8-methoxy-1,2,3,4-tetrahydrochromeno[3,4- c]pyridin-5-one (1), a putative dopamine D 4 receptor antagonist (k i = 8.7 nM), was labeled by positron-emitter ( 11C) and its pharmacological evaluation was carried out with in vitro quantitative autoradiography and positron emission tomography (PET). 11C-Methylation of a corresponding desmethyl precursor (2) with [ 11C]CH 3I gave [ 11C]1 with ≥98% of radiochemical purity after HPLC purification and 67–90 GBq/μmol of specific activity at the end of synthesis. The in vitro autoradiography using rat brain sections demonstrated that [ 11C]1 shows no specific binding to the D 4 receptors, but a high specific binding to sigma 1 receptors (IC 50 = 105 nM). In the PET study with monkey brain, [ 11C]1 was highly taken up by the brain and trapped in the brain for at least 90 min. The distribution pattern of radioactivity in the brain was striatum > thalamus > frontal cortex > cerebellum, which was same as the result of in vitro autoradiography. Pre-treatment with non-radioactive 1 (1 mg/kg) produced a significant reduction of radioactivity in all the regions including the cerebellum. Pre-treatment with (+)pentazocine (1 mg/kg), a selective σ 1 receptor agonist, also reduced the radioactivity in the same regions to a similar extent. These results indicate that [ 11C]1 may have some specific binding to the sigma 1 receptors, which is consistent with the result of in vitro autoradiography.

Dynamic turnover of arabinogalactan proteins in cultured Arabidopsis cells

Arabinogalactan proteins (AGPs) are very large proteoglycans thought to have more of a signaling than a structural role when secreted into the plant cell wall. AGPs are also the first known family of abundant plant proteins synthesized with glycosylphosphatidylinositol(GPI) anchors. Nascent cellular Arabidopsis AGPs, still bearing an intact GPI anchor, and AGPs copiously discharged into the culture medium after phospholipase-cleavage of their anchor were each represented by more than 15 seemingly homologous molecular species of increasing size. In washed cells 3H-ethanolamine was slowly incorporated into each AGP’s GPI anchor via phosphatidylethanolamine. Pulse labeling of AGPs by 3H-acetate and by 3H-galactose was much more rapid, allowing labeled AGP detection in the growth medium within 1 h. HPLC analysis of the radiolabel distribution in AGPs secreted within 1–8 h revealed a sharp preference for the larger molecular species. After several hours a population of smaller radioactive AGP species began to appear in the medium. Following certain manipulations of the cells newly secreted AGP species measured by HPLC on a relative mass basis formed a pattern surprisingly different from the radioactivity pattern, although larger species still dominated. Thus Arabidopsis cells appear capable of releasing higher mass AGP species apparently stored in cell wall sites along with a unique mixture of freshly synthesized AGPs in combinations potentially active in signaling.

Microautoradiographic localisation of a glucosinolate precursor to specific cells in Brassica napus L. embryos indicates a separate transport pathway into myrosin cells.

The in-situ localisation of a desulpho-glucosinolate precursor has been studied by microautoradiography of cryo-sections from immature seeds and pods of the high-glucosinolate Brassica napus L. cv. Argentine collected 23 days after pollination. After feeding with the tritium-labelled glucosinolate precursor [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid, embryo radicles, cotyledons and pod-wall were frozen in liquid nitrogen. Cryotome sections were freeze-dried and coated with nuclear emulsion autoradiographic film. A distinct pattern of radioactivity derived from the glucosinolate precursor was found in specific cells in both radicle and cotyledons. In contrast, the labelling in pod walls was not cell specific, but general at the inner side of the pod wall. The results show that the glucosinolate/desulphoglucosinolate was localised in specific cells, in a pattern resembling that of myrosin cells known to contain myrosinase (EC 3.2.3.1). In addition [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to immature seeds and pods of B. napus and a quantitative incorporation into 2-hydroxy-3-butenylglucosinolate and 3-butenyl-glucosinolate was observed. When [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to 4-day-old seedlings the label was taken up by all tissues. We propose a model in which glucosinolate/desulphoglucosinolates are transported to myrosin cells to participate in the myrosinase-glucosinolate multifunctional defence system.

Differentiating the biosynthesis of pseudomonic acids A and B.

Pseudomonic acid A (1) has been the dominant commercial pseudomonate antibiotic produced by Pseudomonas fluorescens. In specific shaken flask conditions initial fermentation accumulation of 1 is followed by preferential accumulation of the 8-hydroxy derivative, pseudomonic acid B (2). Biosynthetic probing with a pulse of [1-14C] acetate or L-[methyl-14C] methionine at early, mid and late stages of the fermentation gave relative patterns of radioactivity in 1 and 2 that are inconsistent with an assumption that 2 arises by oxidation of 1, or that 1 is formed by reduction of 2. Since [methyl-14C] methionine only labels carbons in the 12-carbon part of the pseudomonate molecule that is thought to be an early biosynthetic moiety, the evidence from radiolabelling experiments implies that preferential early oxidation of this biosynthetic intermediate causes the pathway diversion to accumulate 2 instead of 1.

Oil biosynthesis in microsomal membrane preparations from Mortierella alpina

Microsomal membrane preparations from Mortierella alpina catalysed the conversion of sn-glycerol 3-phosphate and [(14)C]oleoyl-CoA to radioactive phosphatidic acid, diacylglycerol and triacylglycerol. Experiments with lysophosphatidic acid and [(14)C]oleoyl-CoA gave a similar pattern of radioactivity in the complex lipids. The specific activity of lysophosphatidate acyltransferase was almost eight times greater than sn-glycerol-3-phosphate acyltransferase, indicating that the first acylation step was limiting in oil assembly in the microsomal membranes. Little conversion of radioactive oleate into phosphatidylcholine occurred, suggesting that triacylglycerol assembly and its relationship to phosphatidylcholine metabolism differed to that found in oilseeds.

Lipid conversions during enrichment of Artemia

Artemia nauplii were enriched for 24 h with radiolabelled fatty acid ethyl esters and then starved for a subsequent period of 24 h. Analyses of the distribution of radioactivity in lipids from samples taken at the end of the enrichment period and after the subsequent starvation showed that the ethyl esters were readily converted into other lipid classes, mainly triacylglycerols, during assimilation by the nauplii. The proportions of radioactivity recovered in free fatty acids and phospholipids increased during the starvation period indicating the mobilisation of fatty acids from triacylglycerols for use in catabolism and in the formation of biomembrane lipids. The distribution pattern of radioactivity from [U– 14 C ]22:6 n−3 in the fatty acids of the nauplii demonstrates that Artemia are capable of retroconverting 22:6 n−3 to 20:5 n−3.

Synthesis of factor by gastric cancer derived cell lines

Arabinogalactan proteins (AGPs) are very large proteoglycans thought to have more of a signaling than a structural role when secreted into the plant cell wall. AGPs are also the first known family of abundant plant proteins synthesized with glycosylphosphatidylinositol(GPI) anchors. Nascent cellular Arabidopsis AGPs, still bearing an intact GPI anchor, and AGPs copiously discharged into the culture medium after phospholipase-cleavage of their anchor were each represented by more than 15 seemingly homologous molecular species of increasing size. In washed cells 3H-ethanolamine was slowly incorporated into each AGP’s GPI anchor via phosphatidylethanolamine. Pulse labeling of AGPs by 3H-acetate and by 3H-galactose was much more rapid, allowing labeled AGP detection in the growth medium within 1 h. HPLC analysis of the radiolabel distribution in AGPs secreted within 1–8 h revealed a sharp preference for the larger molecular species. After several hours a population of smaller radioactive AGP species began to appear in the medium. Following certain manipulations of the cells newly secreted AGP species measured by HPLC on a relative mass basis formed a pattern surprisingly different from the radioactivity pattern, although larger species still dominated. Thus Arabidopsis cells appear capable of releasing higher mass AGP species apparently stored in cell wall sites along with a unique mixture of freshly synthesized AGPs in combinations potentially active in signaling.

Absorption and distribution kinetics of the major Parietaria judaica allergen (Par j 1) administered by noninjectable routes in healthy human beings

Background and objective: The clinical effectiveness of noninjectable routes for specific immunotherapy has been demonstrated in many studies, but no data are available on the kinetics of allergens administered by these routes. Therefore we studied the kinetics of the radiolabeled purified major Parietaria judaica allergen (Par j 1) after sublingual, oral, and intranasal administration to healthy human beings. Methods: After tracer administration (10 to 12.5 μg of Par j 1 labeled with iodine 123) to nonallergic volunteers, scintigraphic images were recorded at various times. Blood samples were also obtained at serial intervals to evaluate the absorption and distribution of radioactivity in plasma and to identify circulating radioactive species by molecular exclusion gel chromatography. Results: When the sublingual route was used, no circulating radioactivity was detected until the tracer was kept under the tongue. The labeled allergen was rapidly degraded and absorbed in the gastrointestinal tract after swallowing. Plasma radioactivity peaked at about 1.5 to 3 hours and was mostly represented by free radioiodine and small radiolabeled peptides. Some activity not caused by free 123I remained associated with the oral mucosa up to 18 to 20 hours after administration. When the oral route was used, the results were similar to those observed after swallowing the sublingually administered allergen but without any persistence of the tracer in the mouth. When the intranasal route was used, the pattern of plasma radioactivity mimicked that of the sublingual and oral routes, with absorption of activity from the radiolabeled allergen occurring in the gastrointestinal tract after transport to the pharynx by mucociliary cle arance. A relevant fraction of the tracer was retained on the nasal mucosa up to 48 hours after administration. Conclusion: The data in this study provide the first experimental basis for exploring the in vivo kinetics of allergen administered through noninjectable routes for specific immunotherapy in human beings. (J Allergy Clin Immunol 1997;100:122-9.)