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 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.

Reversal learning enhanced by lysergic acid diethylamide (LSD): concomitant rise in brain 5-hydroxytryptamine levels.

1 Small doses of lysergic acid diethylamide (LSD) (12.5-50 mug/kg) consistently facilitated learning of a brightness discrimination reversal.2 2-Bromo-lysergic acid diethylamide (BOL-148), a structural analogue of LSD, with similar peripheral anti-5-hydroxytrypamine activity but no psychotomimetic properties, had no effect in this learning situation at a similar dose (25 mug/kg).3 LSD, but not BOL-148, caused a small but significant increase in brain 5-hydroxytryptamine levels, but had no effect on the levels of catecholamines in the brain at 25 mug/kg.

Studies on the reactivity of acyl glucuronides--I. Phenolic glucuronidation of isomers of diflunisal acyl glucuronide in the rat.

Diflunisal (DF) is metabolized primarily to its acyl glucuronide (DAG), phenolic glucuronide (DPG) and sulphate (DS) conjugates. Whereas DPG and DS are stable at physiological pH, DAG is unstable, undergoing hydrolysis (regeneration of DF) and rearrangement (intramolecular acyl migration to the 2-, 3- and 4-O-acyl-positional isomers). We have compared the in vivo disposition of DAG with that of an equimolar mixture of its three isomers after i.v. administration at 10 mg DF equivalents/kg to conscious, bile-exteriorized rats. After dosing with DAG, excretion in urine and bile (46% as DAG), hydrolysis (as assessed by recovery of 9% DPG and 8% DS resulting from reconjugation of liberated DF) and rearrangement (17% recovery as isomers of DAG) were important pathways. Highly polar metabolites excreted almost exclusively in bile and accounting for 13% of the dose were identified as an approximate 4:1 mixture of the 2- and 3-O-isomers of DAG which had been glucuronidated at the phenolic function of the salicylate ring i.e. "diglucuronides" of DF. Evidence for trace quantities only of the phenolic glucuronides of the 4-O-isomer of DAG, and of DAG itself, was found. After dosing rats with an equimolar mixture of the isomers, 52% was recovered (as the isomers) in urine and bile in 6 hr. Hydrolysis was less important--less than 3% (total) of the dose was recovered as DPG and DS. The phenolic glucuronides of the 2- and 3-O-isomers (ratio ca. 3:7) accounted for 37%. Evidence for appreciable formation of the phenolic glucuronide of the 4-O-isomer was not found. In one rat dosed with DPG, there was no evidence for further glucuronidation of the salicylate ring at its carboxy function. The data suggest that the 2- and 3-O-isomers of DAG, but not the 4-O-isomer, DAG itself or DPG, are good substrates for further glucuronidation.

Studies on the reactivity of acyl glucuronides--II. Interaction of diflunisal acyl glucuronide and its isomers with human serum albumin in vitro.

A major metabolite of diflunisal (DF) is its reactive acyl glucuronide conjugate (DAG) which can undergo hydrolysis (regeneration of DF), intramolecular rearrangement (isomerization via acyl migration) and intermolecular reactions with nucleophiles. We have compared the fate of DAG and its individual 2-, 3- and 4-O-acyl positional isomers (at ca. 55 micrograms DF equivalents/mL) after incubation with human serum albumin (HSA, 40 mg/mL) at pH 7.4 and 37 degrees. Initial half-lives (T1/2) for DAG and its 2-, 3- and 4-isomers were 53, 75, 61 and 26 min, respectively. DAG was more labile to hydrolysis than any of its isomers but the latter, in particular the 4-isomer, were much better substrates for formation of covalent DF-HSA adducts. After a 2-hr incubation, 2.4, 8.2, 13.7 and 36.6% of substrate DAG and its 2-, 3- and 4-isomers (respectively) were present as DF-HSA adducts. With long term incubation, the concentrations of adducts so generated in situ declined in a biphasic manner, with apparent terminal T1/2 values of ca. 28 days. DAG was much more labile to transacylation with methanol (i.e. formation of DF methyl ester) than an equimolar mixture of its isomers after incubation in a 1:1 methanol:pH 7.4 buffer solution at 37 degrees (T1/2 values of 5 and 70 min, respectively). The data do not support direct transacylation with nucleophilic groups on protein as the predominant mechanism of formation of covalent DF-HSA adducts in vitro.

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 reactivity of acyl glucuronides--IV. Covalent binding of diflunisal to tissues of the rat.

Acyl glucuronides have been shown to be reactive electrophilic metabolites capable of undergoing hydrolysis, rearrangement (isomerization via acyl migration) and covalent binding reactions to plasma protein. The present study was undertaken to explore the occurrence and extent of in vivo formation of covalent adducts of diflunisal (DF), a salicylate derivative which forms a reactive acyl glucuronide, with tissues and plasma protein of rats. Groups of rats were given 50 mg DF/kg i.v. twice daily for periods of up to 7 days. Steady state plasma concentrations of reversibly bound DF and its conjugates (as measured 6 hr after a dose) were achieved by the third day of dosing. T 1/2 values after cessation of dosing were about 5-10 hr. By contrast, covalent DF-tissue adducts steadily accumulated over the 7-day dosing period. Maximum concentrations, measured 6 hr after the last dose, were 4.8 (liver), 1.0 (kidney), 0.74 (plasma), 0.26 (small intestine minus contents), 0.27 (large intestine minus contents) and 0.20 (skeletal muscle) microgram DF/g tissue or/mL plasma. T 1/2 values of about 50, 67, 18, 38 and 43 hr were obtained for liver, kidney, plasma and small and large intestine (respectively) after cessation of dosing. Thus, the study of acyl glucuronide reactivity and the question of any derived toxicity or immune responses should consider the formation of long-lived adducts in tissues as well as in plasma.

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.

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.

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 benign gonadal stromal tumor of the testis of spindle fibroblastic type.

Non-Leydig cell gonadal stromal tumors of the testis are rare and most are benign. Criteria for determining malignancy are poorly defined. A gonadal stromal tumor of spindle fibroblastic cells presented in a 34 year old male. The patient remains alive and well with no evidence of metastasis 3 years following surgery. Light and electron microscopical features of the tumor are described.

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.

Cardiac rupture: 30 consecutive cases from a series of medicolegal autopsies.

Factors underlying spontaneous cardiac rupture were studied in a consecutive series of 30 hearts with ruptured infarcts removed at medicolegal autopsy. Normal and diseased heart muscle and narrowed coronary arteries were examined microscopically. The average age of the 15 women at death was 80 years, and of the 15 men, 73 years. All of the ruptures occurred through a recent transmural left ventricular myocardial infarction which was associated with coronary arteries severely narrowed by atherosclerosis. Nine (33 percent) of the cases showed an occlusive coronary thrombosis. Many of the subjects had no symptoms of recent myocardial ischaemia.

Scotopic thresholds of rhesus monkeys at different grating densities under normal and low-protein diets.

Scotopic thresholds of rhesus monkeys were determined over a range of luminance levels, from 1138 to 17.8 x 10(-9) cd/cm2, and grating densities, from .132 to 1.58 lines per cm. The effects of a low-protein diet on these thresholds were also investigated, and standard stimulus objects within the discrimination learning situation were employed. Obtained thresholds decreased systematically from about 350 to 20 x 10(-9) cd/cm2, and there were no significant differences due to dietary protein level.

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.

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.

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.