Management of radioactive waste gases from PET radiopharmaceutical synthesis using cost effective capture systems integrated with a cyclotron safety system.

The emphasis on the reduction of gaseous radioactive effluent associated with PET radiochemistry laboratories has increased. Various radioactive gas capture strategies have been employed historically including expensive automated compression systems. We have implemented a new cost-effective strategy employing gas capture bags with electronic feedback that are integrated with the cyclotron safety system. Our strategy is suitable for multiple automated 18F radiosynthesis modules and individual automated 11C radiosynthesis modules. We describe novel gas capture systems that minimize the risk of human error and are routinely used in our facility.

A critical cysteine residue in monoacylglycerol lipase is targeted by a new class of isothiazolinone-based enzyme inhibitors.

BACKGROUND AND PURPOSE: Monoacylglycerol lipase (MGL) is a presynaptic serine hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine residue 208. EXPERIMENTAL APPROACH: A series of cysteine traps were screened for the ability to inhibit MGL in vitro. Rapid dilution assays were performed to determine reversibility of inhibition. Molecular modelling and site-directed mutagenesis were utilized to identify cysteine residues targeted by the inhibitors. KEY RESULTS: The screening revealed that 2-octyl-4-isothiazolin-3-one (octhilinone) inhibited purified rat recombinant MGL (IC(50)= 88 +/- 12 nM) through a partially reversible mechanism. Initial structure-activity relationship studies showed that substitution of the n-octyl group of octhilinone with a more lipophilic oleoyl group increased inhibitor potency (IC(50)= 43 +/- 8 nM), while substitution with a methyl group produced the opposite effect (IC(50)= 239 +/- 68 nM). The inhibitory potency of octhilinone was selectively decreased by mutating cysteine 208 in MGL to glycine (IC(50); wild-type, 151 +/- 17 nM; C208G, 722 +/- 74 nM), but not by mutation of other cysteine residues (C32, C55, C201, C208 and C242). CONCLUSIONS AND IMPLICATIONS: The results indicated that cysteine 208 plays an important role in MGL function and identified a novel class of isothiazolinone-based MGL inhibitors with nanomolar potency in vitro.

Conjugation of desmethylnaproxen in the rat--a novel acyl glucuronide-sulfate diconjugate as a major biliary metabolite.

The nonsteroidal anti-inflammatory drug naproxen is primarily metabolized in humans by acyl glucuronidation to form naproxen acyl glucuronide and by O-dealkylation to form 6-O-desmethylnaproxen (DMN). DMN contains both carboxy and phenolic groups and has been shown to form acyl glucuronide and sulfate conjugates. This project aimed to investigate whether DMN formed a phenolic glucuronide and diglucuronide(s) (with both the carboxy and phenolic groups glucuronidated). Male Sprague-Dawley rats (300-350 g) with exteriorized bile flow were dosed i.v. with DMN at 50 mg/kg. Four major DMN-related peaks were detected in bile by high-performance liquid chromatography (HPLC) analysis at 225 nm, including the known acyl glucuronide and sulfate conjugates. Selective hydrolyses using acidic and alkaline conditions and digestion with beta-glucuronidase allowed tentative identification of the two unknown peaks as the phenolic glucuronide of DMN and a novel acyl glucuronide-sulfate diconjugate of DMN (i.e., formed by sulfonation of the phenolic group and glucuronidation of the carboxy group). The identities were confirmed by liquid chromatography-tandem mass spectrometry analysis of individual HPLC fractions. Total recovery of the DMN dose was approximately 80%, with the sulfate conjugate (50%) and unchanged DMN (10%) being excreted predominantly in urine and the acyl glucuronide (10%), phenolic glucuronide (6%), and acyl glucuronide-sulfate diconjugate (4%) being excreted predominantly or exclusively in bile. No evidence for a diglucuronide metabolite of DMN was found in either bile or urine of the DMN-dosed rats.

Rearrangement of diflunisal acyl glucuronide into its beta-glucuronidase-resistant isomers facilitates transport through the small intestine to the colon of the rat.

Many non-steroidal anti-inflammatory drugs (NSAIDs) which form acyl glucuronide conjugates as major metabolites have shown an antiproliferative effect on colorectal tumors. This study assesses the extent to which rearrangement of an acyl glucuronide metabolite of a model NSAID into beta-glucuronidase-resistant isomers facilitates its passage through the small intestine to reach the colon. Rats were dosed orally with diflunisal (DF), its acyl glucuronide (DAG) and a mixture of rearrangement isomers (iso-DAG) at 10 mg DF equivalents/kg. The parent drug DF appeared in plasma after all doses, with maximum concentrations of 20.5+/-2.5, 28.8+/-8.3 and 11.0+/-1.6 microg DF/ml respectively, obtained at 3.8+/-0.3, 3.6+/-1.8 and 7.5+/-0.9 hr after the DF, DAG and iso-DAG doses respectively. At 48 hr, 16.2+/-3.3, 19.8+/-0.8 and 42.9+/-10.1% of the doses respectively were recovered in feces, with < or = 1% remaining in the intestine. About half of each dose was recovered as DF and metabolites in 48 hr urine: for DF and DAG doses, the majority was in the first 24 hr urine, whereas for iso-DAG doses, recoveries in the first and second 24 hr periods were similar. The results show that hydrolysis of both DAG and iso-DAG, and absorption of liberated DF, occur during passage through the gut, but that these processes occur more slowly and to a lesser degree for iso-DAG. The intrinsic hydrolytic capacities of various intestinal segments (including contents) towards DAG and iso-DAG were obtained by incubating homogenates under saturating concentrations of DAG/iso-DAG at 37 degrees C. Upper small intestine, lower small intestine, caecum and colon released 2400, 3200, 9200 and 22800 microg DF/hr/g tissue plus contents respectively from DAG substrate, and 18, 10, 140 and 120 microg DF/hr/g tissue plus contents respectively from iso-DAG substrate. The much greater resistance of iso-DAG to hydrolysis appears attributable to its resistance to beta-glucuronidases. The data suggest that in rats dosed with DF, DAG excreted in bile would be substantially hydrolysed in the small intestine and liberated DF reabsorbed, but that portion which rearranges to iso-DAG would likely reach the colon.

A cell kinetic analysis of human umbilical vein endothelial cells.

Cultures of normal human cells 'age' and become senescent in vitro due to a continuously declining mitotic fraction. Although endothelial cells represent a tissue of major relevance in the development of age-related vascular disease, the rate at which these cells senesce has never been systematically measured in culture. Accordingly the population kinetics of human vascular endothelial cells (HUVECs) serially passaged in vitro has been studied in order to determine (i) the rate of decline in the growth fraction; (ii) the rate of increase of the senescent fraction and (iii) the relationship between changes in these parameters and the baseline rate of apoptosis. Immunocytochemical visualisation of the growth fraction using antisera to the proliferation marker pKi67 showed a rate of decline in the growth fraction of 4.43+/-0.31% per population doubling. This was not accompanied by any change in cell cycle time as assessed using time lapse video microscopy. The number of senescent cells within the population increased at a rate of 6.47+/-0.3% as assessed by senescence associated beta-galactosidase activity. The baseline rate of apoptosis as measured by TUNEL remained essentially unchanged (0.31+/-0.07%) during this process. These data show (i) that senescence and apoptosis are unrelated processes in HUVEC and (ii) that senescent cells rapidly and progressively accumulate in dividing populations of endothelial cells. The physiological relevance of these observations is discussed.

Computer models for masked hearing experiments with beluga whales (Delphinapterus leucas).

Environmental assessments of manmade noise and its effects on marine mammals need to address the question of how noise interferes with animal vocalizations. Seeking the answer with animal experiments is very time consuming, costly, and often infeasible. This article examines the possibility of estimating results with software models. A matched filter, spectrogram cross-correlation, critical band cross-correlation, and a back-propagation neural network detected a beluga vocalization in three types of ocean noise. Performance was compared to masked hearing experiments with a beluga whale [C. Erbe and D. M. Farmer, Deep-Sea Res. II 45, 1373-1388 (1998)]. The artificial neural network simulated the animal data most closely and raised confidence in its ability to predict the interference of a variety of noise source with a variety of vocalizations.

Systemic administration of amylin increases bone mass, linear growth, and adiposity in adult male mice.

Amylin is a peptide hormone cosecreted with insulin from the pancreatic beta-cells that can act as an osteoblast mitogen and as an inhibitor of bone resorption. The effects on bone of its systemic administration are uncertain. The present study addresses this question in adult male mice that were given daily subcutaneous injections of amylin (10.5 microgram) or vehicle (n = 20 in each group) for 4 wk. Histomorphometric indices of bone formation increased 30-100% in the amylin-treated group, whereas resorption indices were reduced by approximately 70% (P < 0.005 for all indices). Total bone volume in the proximal tibia was 13.5 +/- 1.4% in control animals and 23.0 +/- 2.0% in those receiving amylin (P = 0.0005). Cortical width, tibial growth plate width, tibial length, body weight, and fat mass were all increased in the amylin-treated group. It is concluded that systemic administration of amylin increases skeletal mass and linear bone growth. This peptide has potential as a therapy for osteoporosis if its bone effects can be dissociated from those on soft tissue mass.

Implementation of image-guided large-core needle biopsy of the breast on a limited budget.

Image-guided large-core needle biopsy (LCNB) of the breast is becoming widely accepted as an accurate, minimally invasive and economical way to obtain a tissue diagnosis of breast lesions. However, much of this work has been done with expensive dedicated equipment, often favouring stereotaxic guidance. Image-guided LCNB was introduced to Middlemore Hospital based around existing inexpensive equipment, and stereotaxic or ultrasound guidance was chosen depending on which showed the lesion best. Multidisciplinary clinical, radiological and pathological assessment of each case was performed. The results of 213 biopsies (112 stereotaxic and 101 ultrasound guidance) are reported here. Malignancy was shown or suspected in 85 biopsies (40%). The absolute sensitivity for malignancy was 97% with complete sensitivity of 100%. The absolute specificity was 100% and the complete specificity 98.5%. Large-core needle biopsy can be successfully implemented in a large institution without investment in expensive equipment while maintaining high ratios of malignant/benign biopsies. Attention to technique and careful multidisciplinary review are important to the success of such a programme.

Adenoviral expression of murine serum amyloid A proteins to study amyloid fibrillogenesis.

Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro and in vivo.

Perforated diverticulum of the ascending colon: pre-operative diagnosis by ultrasound.

A case of acute perforation of a diverticulum of the ascending colon is presented. The ultrasound features that helped in making a pre-operative diagnosis are discussed. This rare surgical emergency and the similar clinical entities of perforated caecal or transverse colon diverticula are often clinically misdiagnosed as more common conditions such as acute appendicitis, acute cholecystitis or perforated ulcer. Awareness of this entity will help radiologists in making a correct pre-operative diagnosis.

The utility of the bile-exteriorized rat as a source of reactive acyl glucuronides: studies with zomepirac.

Acyl glucuronide conjugates of acidic drugs are chemically unstable metabolites, able to undergo a number of reactions including covalent binding interactions with proteins. The question of whether any toxicological or immunological responses result from such covalent modification of native proteins in vivo is topical. Study of acyl glucuronide reactivity thus requires a convenient source of these metabolites. The utility of the bile-exteriorized rat for this purpose is highlighted herein using the formerly marketed nonsteroidal antiinflammatory agent zomepirac. Zomepirac was injected i.v. at 60 mg/kg four times into bile-exteriorized rats at 6-h intervals. The 24-h bile samples contained ca. 24% of zomepirac doses as zomepirac acyl glucuronide (ZAG). Purification was achieved by washing of the acidified bile with etherhexane, extraction into ethyl acetate, semipreparative HPLC, and crystallization. Overall recovery through the purification procedure was ca. 50%. Identity as ZAG was confirmed by mass spectrometry. The approach takes advantage of the robust glucuronidation capacity of the rat, especially at higher drug doses, and of its ability to preferentially excrete hepatically formed drug glucuronides into bile rather than into urine via blood. Prior to this work, ZAG was presumed to be only a minor metabolite of zomepirac in rats, based on early urinary recovery studies. Thus, measurement of urinary acyl glucuronide conjugates in the rat may severely underestimate their true formation in this species.

Vesico-hepato-renal cycling of acidic drugs via their reactive acyl glucuronide metabolites? Studies with diflunisal in rats.

1. Deconjugation-reconjugation cycling of acidic drugs is known to occur in vivo via the hydrolysis of their reactive acyl glucuronide metabolites during their circulation in the blood (systemic cycling) or during their passage through the gut after biliary excretion (enterohepatic cycling). Whether such cycling occurs after renal excretion via hydrolysis in the urinary bladder followed by absorption of liberated drug (vesico-hepato-renal cycling) was investigated in rats using diflunisal (DF) and its acyl glucuronide (DFAG) as model compounds. 2. After administration of DF (1 mg/0.5 mL buffer, pH 7) into the bladder of anaesthetized bile-exteriorized rats, DF appeared rapidly in plasma, achieving peak concentrations of 7 micrograms/mL at 1 h. At 4 h, 30% of the dose was recovered as metabolites, mainly DFAG and DF phenolic glucuronide (DFPG) in bile, while 30% was recovered as unchanged DF from the bladder. 3. By contrast, after intravesical administration of an equimolar amount of DFAG at pH 7 or 5, DFAG itself was not detectable in plasma. Plasma concentrations of DF were barely detectable, with only approximately 1% of the administered dose recovered as metabolites in bile. 4. The data thus show that, although DF itself undergoes facile absorption from the urinary bladder of healthy rats, vesico-hepato-renal cycling of DF via DFAG appears to be of only minor quantitative importance.

High-dose pamidronate in the management of resistant Paget's disease.

Current recommendations for the total dose of intravenous pamidronate to be used in the treatment of Paget's disease range up to 400 mg per course, although up to 980 mg has been suggested for resistant cases. However, in a proportion of Paget's disease patients remission is difficult to induce and maintain. In five patients with resistant symptomatic Paget's disease, in whom a variety of antipagetic therapies had failed to induce remission, we have examined the effects of high dose pamidronate (1.44-2.52 g intravenously over 12-42 weeks). All five subjects had a marked symptomatic improvement, and disease activity was suppressed to a greater extent than had been achieved previously, but in only one did alkaline phosphatase activity suppress into the normal range. A plateau in the biochemical response was evident, with successive pamidronate doses of 120 mg producing smaller decrements in alkaline phosphatase. The plateau was reproducible on repeated courses. Bone biopsies in two patients showed continued pagetic activity with an increased mineralization rate and no osteomalacia. Worthwhile clinical and biochemical improvements can be obtained in patients with resistant Paget's disease by the use of high-dose pamidronate. Though this approach does not seem to cause defective mineralization, it may be difficult to suppress disease activity completely.

Association of apolipoprotein E with murine amyloid A protein amyloid.

BACKGROUND: Experimental amyloid A protein (AA) amyloidosis in mice is the most rapid type of amyloid formed, thus providing a valuable model to study amyloid formation. Recent studies have suggested the importance of apolipoprotein E (apoE) in Alzheimer's disease and systemic amyloidoses. To help understand the role of apoE in amyloidoses, we examined amyloid tissue for the presence of apoE in mouse AA amyloid. EXPERIMENTAL DESIGN: Mice were injected with amyloid-enhancing factor and silver nitrate to induce amyloid deposition. Spleens were examined for Congo red staining and serum amyloid A, serum amyloid P component, and apoE immunostaining. In addition, RNA analysis was performed to measure the expression of apoE in various tissues after amyloid induction. RESULTS: We have found that apoE is associated with mouse amyloid. Ab to apoE consistently detected the presence of increased levels of apoE in amyloid tissue. Immunohistochemical analyses confirmed that the apoE immunoreactivity co-associated with AA and serum amyloid P in the amyloid fibrils. Northern blot analysis of amyloid tissue showed an increase in apoE messenger RNA compared with control tissue. CONCLUSIONS: This is the first demonstration of apoE in mouse amyloid tissue. The data presented here, along with previous studies, suggest that apoE may be involved in amyloidogenesis. These studies validate the mouse model for studying the role of apoE in amyloid fibrillogenesis. The use of transgenic and gene-inactivated mice will help to elucidate the role and mechanism of apoE in amyloid formation.

Characterization and formation of the glutathione conjugate of clofibric acid.

The incubation of 1-O-clofibryl glucuronide (1-O-CAG), a metabolite of clofibrate, with glutathione (GSH) resulted in the appearance of a new peak when analyzed by HPLC. The use of HPLC coupled to electrospray-MS permitted the identification of the peak as S-(p-chlorophenoxy-2-methylpropanoyl)glutathione (CA-SG), formed by nucleophilic displacement of the glucuronide moiety from 1-O-CAG. Conjugate formation was enhanced 8-fold by rat liver glutathione S-transferases (GSTs). GSH was unreactive with isomers of 1-O-CAG formed by acyl migration, indicating that 1-O-CAG itself was the preferred substrate. Rearrangement of 1-O-CAG to its isomers in vitro, was found to be decreased in the presence of GSH. In vivo studies indicated that, following an intravenous infusion of clofibric acid to rats (75 mg/kg), the concentration of CA-SG excreted in bile over 4 hr, was approximately 0.1% of the concurrent CAG concentrations. Although these results indicate a minor role for GST-catalyzed reactions in clofibrate metabolism in vivo, they do define 1-O-acyl-linked glucuronides as a new class of substrates for GSTs.

Excretion of 3-hydroxy-diflunisal as a monosulphate conjugate--identification using ESI-MS.

Electrospray-ionization mass spectrometry was used to identify a novel, highly polar metabolite of diflunisal isolated from Gunn rat urine. Negative ion spectra were obtained of the sulphate conjugate of diflunisal and the new metabolite, which was identified as a sulphate conjugate of 3-hydroxydiflunisal.

Studies on the reactivity of acyl glucuronides--VII. Salicyl acyl glucuronide reactivity in vitro and covalent binding of salicylic acid to plasma protein of humans taking aspirin.

Salicyl acyl glucuronide (SAG) is a significant metabolite of salicylic acid (SA) and aspirin. We have shown that, under physiological conditions in vitro, SAG undergoes rearrangement in a manner consistent with acyl migration to its 2-, 3- and 4-O-acyl positional isomers as the predominant pathway (T1/2 values were 1.4-1.7 hr in buffer at pH 7.4 and 37 degrees). Incubation of SAG or a mixture of its rearrangement isomers (iso-SAG) (each at approximately 50 micrograms SA equivalents/mL) with human serum albumin (HSA, at approximately 40 mg/mL) revealed the formation of covalent adducts with the protein, with peak concentrations of 1-2 micrograms SA equivalents/mL. The data support a role for the rearrangement/glycation mechanism of adduct formation. Covalent adducts of SA were also detected in the plasma of humans taking aspirin (at > or = 1200 mg/day), but the concentrations were low (<< 100 ng SA equivalents/mL). Reactivity of SAG thus provides a mechanism (though of uncertain quantitative importance) of covalent attachment of the salicyl moiety of aspirin to tissue macromolecules, which is in addition to its well-known acetylating capacity.

Studies on the reactivity of acyl glucuronides--V. Glucuronide-derived covalent binding of diflunisal to bladder tissue of rats and its modulation by urinary pH and beta-glucuronidase.

Acyl glucuronide conjugates of acidic drugs have been shown to be reactive metabolites capable of undergoing non-enzymic hydrolysis, rearrangement (isomerization via acyl migration) and covalent binding reactions with plasma protein. In an earlier study (King and Dickinson, Biochem Pharmacol 45: 1043-1047, 1993), we documented formation of covalent adducts of diflunisal (DF), a salicylate derivative which is metabolized in part to a reactive acyl glucuronide (DAG), with liver, kidney, skeletal muscle and small and large intestine (in addition to plasma protein) of rats given the drug i.v. twice daily at 50 mg DF/kg for 7 days. The present study shows that covalent adducts of DF were also formed with urinary bladder tissue of these rats, achieving concentrations (ca. 5 micrograms DF equivalents/g tissue) higher than those found in the other tissues noted above. After cessation of dosing, the adduct concentrations declined with an apparent T 1/2 value of ca. 20 hr. Adducts were also formed ex vivo in excised rat bladders in which DAG or a prepared mixture of its acyl migration isomers (iso-DAG) were incubated at pH 5.0, 6.5 and 8.0. After 8 hr incubation, the highest concentrations (ca. 11 micrograms DF equivalents/g) were produced with iso-DAG at pH 5.0, and the lowest (ca. 2.3 micrograms DF equivalents/g) with DAG at pH 5.0. However, a major competing reaction for DAG (at least at pH 5.0) was hydrolysis by beta-glucuronidases originating from bladder tissue. By contrast, iso-DAG was quite resistant to such hydrolysis. The phenolic glucuronide conjugate, another important metabolite of DF, was hydrolysed only slowly. Similar results were obtained in fresh rat urine adjusted to pH 5.0. The results support covalent DF adduct formation in rat bladder originating from both DAG and iso-DAG as ultimate reactants, though the extent of binding is modulated by both urinary pH and beta-glucuronidases.

Studies on the renal excretion of the acyl glucuronide, phenolic glucuronide and sulphate conjugates of diflunisal.

1. In five healthy male volunteers given multiple doses of diflunisal (DF), renal clearances (CLR) of the acyl glucuronide (DAG), phenolic glucuronide (DPG) and sulphate (DS) conjugates were about 42, 25 and 13 ml min-1, respectively. 2. These relatively low CLR values are probably due largely to the very high plasma protein binding of the conjugates, found in vitro to be 99.0%, 97.8% and 99.45%, respectively. 3. Thus glomerular filtration plays the minor and active tubular secretion the major role in renal excretion of the three conjugates. 4. This conclusion was supported by the effect of probenecid co-administration, which decreased CLR of DAG and DPG by about 70%. CLR for DS could not be calculated when probenecid was co-administered (because of interference by probenecid metabolites in the analysis of DS in urine). 5. Water-induced diuresis had no effect on CLR of the DF conjugates, consistent with tubular reabsorption being negligible.