Automated production at the curie level of no-carrier-added 6-[(18)F]fluoro-L-dopa and 2-[(18)F]fluoro-L-tyrosine on a FASTlab synthesizer.

An efficient, fully automated, enantioselective multi-step synthesis of no-carrier-added (nca) 6-[(18)F]fluoro-L-dopa ([(18)F]FDOPA) and 2-[(18)F]fluoro-L-tyrosine ([(18)F]FTYR) on a GE FASTlab synthesizer in conjunction with an additional high- performance liquid chromatography (HPLC) purification has been developed. A PTC (phase-transfer catalyst) strategy was used to synthesize these two important radiopharmaceuticals. According to recent chemistry improvements, automation of the whole process was implemented in a commercially available GE FASTlab module, with slight hardware modification using single use cassettes and stand-alone HPLC. [(18)F]FDOPA and [(18)F]FTYR were produced in 36.3 ± 3.0% (n = 8) and 50.5 ± 2.7% (n = 10) FASTlab radiochemical yield (decay corrected). The automated radiosynthesis on the FASTlab module requires about 52 min. Total synthesis time including HPLC purification and formulation was about 62 min. Enantiomeric excesses for these two aromatic amino acids were always >95%, and the specific activity of was >740 GBq/µmol. This automated synthesis provides high amount of [(18)F]FDOPA and [(18)F]FTYR (>37 GBq end of synthesis (EOS)). The process, fully adaptable for reliable production across multiple PET sites, could be readily implemented into a clinical good manufacturing process (GMP) environment.

On the use of positron counting for radio-Assay in nuclear pharmaceutical production.

Current techniques for the measurement of radioactivity at various points during PET radiopharmaceutical production and R&D are based on the detection of the annihilation gamma rays from the radionuclide in the labelled compound. The detection systems to measure these gamma rays are usually variations of NaI or CsF scintillation based systems requiring costly and heavy lead shielding to reduce background noise. These detectors inherently suffer from low detection efficiency, high background noise and very poor linearity. They are also unable to provide any reasonably useful position information. A novel positron counting technique is proposed for the radioactivity assay during radiopharmaceutical manufacturing that overcomes these limitations. Detection of positrons instead of gammas offers an unprecedented level of position resolution of the radiation source (down to sub-mm) thanks to the nature of the positron interaction with matter. Counting capability instead of charge integration in the detector brings the sensitivity down to the statistical limits at the same time as offering very high dynamic range and linearity from zero to any arbitrarily high activity. This paper reports on a quantitative comparison between conventional detector systems and the proposed positron counting detector.

Oxidative metabolism of Polytron versus Nagarse mitochondria in hearts of genetically diabetic mice.

We have shown previously that heart mitochondria obtained by the Nagarse method from genetically diabetic mice (C57BL/KsJ db/db) exhibit a defect in oxidizing NAD+-linked substrates (Kuo, T.H., Moore, K.H., Giacomelli, F. and Wiener, J. (1983) Diabetes 32, 781-787). In this study, the oxidative phosphorylation characteristics of cardiac mitochondria isolated by the Polytron method were compared with that of Nagarse mitochondria. Evidence is presented here that in the diabetic heart both Nagarse and Polytron mitochondria have defective pyruvate oxidation, whereas only the former exhibit impaired fatty acid oxidation. Assay of two rate-limiting beta-oxidation enzymes, namely beta-hydroxyacyl-CoA dehydrogenase and beta-ketothiolase, indicates no alteration in specific activities from diabetic mice vs. controls. The data suggest that two populations of mitochondria are present in myocardium and that the defective oxidative metabolism in the cardiac mitochondria of db/db mice may be linked to deficiencies in total NAD + NADH content.

Purification and characterization of two distinct Ca2+-activated proteinases from hearts of hypertensive rats.

Two distinct Ca2+-activated proteinases were purified and characterized from hearts of hypertensive rats. Ca2+-activated proteinases I and II, having low and high Ca2+ requirements, respectively, were first separated by DEAE-cellulose chromatography. The enzymes were then purified individually by different column procedures: chromatography on phenyl-Sepharose, then Sephadex G-200 for proteinase I and reactive-red agarose for proteinase II. The apparent molecular weight of purified proteinase I was 125 000 and that for purified proteinase II was 110 000. Both enzymes are heterodimers made up of a larger catalytic subunit and a smaller subunit devoid of proteinase activity. Ca2+ concentrations for half-maximal activation were 5 microM for proteinase I and 200 microM for proteinase II. Both enzymes were inhibited by sulfhydryl-modifying agents, but exhibited different characteristics in the auto-digestion reaction in the presence of Ca2+. Proteinases I and II were also purified from hearts of normotensive rats and shown to be identical to their respective counterparts from hearts of hypertensive rats. However, proteinase II activity in hypertensive rat hearts was significantly elevated as compared to controls.

Pyruvate dehydrogenase activity in cardiac mitochondria from genetically diabetic mice.

Pyruvate dehydrogenase (PDH) was studied in isolated heart mitochondria from genetically diabetic mice (C57BL/KsJ db/db) with progressive cardiomyopathy. Both the basal activity (active enzyme) and the total activity (the sum of active and inactive enzyme) were determined. In mitochondrial extracts from 8-18-wk-old db/db mice, there was a 73% decrease of basal activity accompanied by a 38% decrease of total activity as compared with controls. The lower basal activity at 8 wk of age suggests an increased conversion of the enzyme into the phosphorylated (inactive) form. Evidence is also given that the conversion of inactive (phosphorylated) enzyme into active (dephosphorylated) enzyme is inhibited in cardiac mitochondria prepared from 8-wk and older db/db mice. These changes coincide with the onset of defective oxidative metabolism and can explain the depressed pyruvate oxidation reported previously.

Defective oxidative metabolism of heart mitochondria from genetically diabetic mice.

Long chain saturated beta-hydroxy fatty acid content and oxidative metabolism were studied in hearts of diabetic mice (C57BL/KsJ db/db) with a progressive cardiomyopathy at intervals of 7, 10, 16, and 26 wk of age. Total beta-hydroxy fatty acid (BHFA) content increases progressively with age in diabetic hearts with a mean value of 143.5 nmol/g dry wt as compared with a mean of 59.6 nmol/g dry wt in control hearts. There was also a redistribution of BHFA in myocardium of diabetic mice when compared with controls, with a relative decrease in beta-hydroxymyristate and an increase of beta-hydroxypalmitate. Oxidative phosphorylation studies using isolated mitochondria from diabetic mice demonstrated depressed state 3 oxidation rates with both palmityl carnitine and pyruvate as substrates. While mitochondrial NADH-oxidase activity was not statistically different from that of controls, there was a significant decrease in mitochondrial total NAD + NADH content in diabetic hearts. In addition, treatment of myocardial tissue with lanthanum demonstrated an abnormal permeability of sarcolemmal, intercalated disc as well as mitochondrial membranes in myocytes of diabetic mice. The data indicate that deficiencies in total NAD + NADH content can account for the depressed state 3 oxidation of palmitylcarnitine and pyruvate in diabetic mice that in turn may explain the abnormal accumulation of BFHA. The latter could play a role in altering the permeability of cardiac cell membranes.

Biodistribution and Radiation Dosimetry for the Novel SV2A Radiotracer [(18)F]UCB-H: First-in-Human Study.

PURPOSE: [(18)F]UCB-H is a novel radiotracer with a high affinity for synaptic vesicle glycoprotein 2A (SV2A), a protein expressed in synaptic vesicles. SV2A is the binding site of levetiracetam, a "first-in-class" antiepileptic drug with a distinct but still poorly understood mechanism of action. The objective of this study was to determine the biodistribution and radiation dosimetry of [(18)F]UCB-H in a human clinical trial and to establish injection limits according to biomedical research guidelines. Additionally, the clinical radiation dosimetry results were compared to estimations in previously published preclinical data. PROCEDURES: Dynamic whole body positron emission tomography/X-ray computed tomography (PET/CT) imaging was performed over approximately 110 min on five healthy male volunteers after injection of 144.5 ± 7.1 MBq (range, 139.1-156.5 MBq) of [(18)F]UCB-H. Major organs were delineated on CT images, and time-activity curves were obtained from co-registered dynamic PET emission scans. The bladder could only be delineated on PET images. Time-integrated activity coefficients were calculated as area under the curve using trapezoidal numerical integration. Urinary excretion data based on PET activities including voiding was also simulated using the dynamic bladder module of OLINDA/EXM. The radiation dosimetry was calculated using OLINDA/EXM. RESULTS: The effective dose to the OLINDA/EXM 70-kg standard male was 1.54 × 10(-2) ± 6.84 × 10(-4) millisieverts (mSv)/MBq, with urinary bladder wall, gallbladder wall, and the liver receiving the highest absorbed dose. The brain, the tracer's main organ of interest, received an absorbed dose of 1.89 × 10(-2) ± 2.32 × 10(-3) mGy/MBq. CONCLUSIONS: This first human dosimetry study of [(18)F]UCB-H indicated that the tracer shows similar radiation burdens to widely used common clinical tracers. Single injections of at maximum 672 MBq for US practice and 649 MBq for European practice keep radiation exposure below recommended limits. Recently published preclinical dosimetry data extrapolated from mice provided satisfactory prediction of total body and effective dose but showed significant differences in organ absorbed doses compared to human data.

Laparoscopic Italian experience with the Lap-Band.

BACKGROUND: An increasing number of surgeons with different levels of experience with laparoscopic surgery and open obesity surgery have started to perform laparoscopic implantation of the Lap-Band. METHODS: An electronic patient data sheet was created and was mailed and e-mailed to all surgeons performing laparoscopic adjustable silicone gastric banding (LASGB) in Italy. Patients were recruited since January 1996. Data on 1,265 Lap-Band System operated patients (258 M/1,007 F; mean BMI 44.1, range 27.0-78.1; mean age 38, range 17-74 years) were collected from 23 surgeons performing this operation. RESULTS: Intra-operative mortality was absent. Post-operative mortality was 0.55% (7 patients) for causes not specifically related to LASGB implantation. The laparotomic conversion rate was 1.7% (22 patients). LASGB related complications occurred in 143 patients (11.3%). Pouch dilatation was diagnosed in 65 (5.2%), and 28 (2.2%) of these underwent re-operation. Band erosion was observed in 24 patients (1.9%). Port or connecting tube-port complications occurred in 54 patients (4.2%), 12 of whom required revision under general anesthesia. Follow-up was obtained at 6, 12, 18, 24, 36 and 48 months, and mean BMI was respectively 38.4, 35.1, 33.1, 30.2, 32.1 and 31.5. The percentage of patients observed at each follow-up was > 60%. There was no intra-operative mortality and no complication-related mortality, with acceptable weight loss. CONCLUSION: The LASGB operation is safe and effective, and deserves wider use for treatment of morbid obesity.

Transcriptional regulation of calcium-activated neutral protease in cardiomyocytes of hypertensive rats.

Increased enzymatic activity of calcium-activated neutral protease II (CANP II) has been reported previously in cardiac tissues of rats with 2 kidney, 1 clip Goldblatt hypertension (2K, 1C-HT); this was associated with elevated intracellular free Ca(++). Because it was suggested that increased levels of the enzyme were responsible for the enhanced activity, CANP II mRNA expression was assessed in cardiomyocytes isolated from 2K, 1C-HT rats and from a genetic model of hypertension, the spontaneously hypertensive rat (SHR). Utilizing a rabbit probe for the large subunit of CANP II (pLM 1006 cDNA), a 3.7 kilobase mRNA band was visualized in Northern blots of poly A+ RNA. Densitometric analysis of the blots revealed that there was a significant (p < 0.005) increase in the levels of CANP II large subunit mRNA in cardiomyocytes of 2K, 1C-HT rats when compared with controls. Interestingly, CANP II mRNA levels were comparable in cardiomyocytes of SHR and Wistar Kyoto (WKY) rats. Results of nuclear runoff transcription assays indicated that enhanced expression of CANP II mRNA in 2K, 1C-HT rat hearts was regulated at the transcriptional level. The data support specific CANP II gene activation in the hearts of renal hypertensive rats.

Selective expression of c-mas proto-oncogene in rat cerebral endothelial cells.

Northern blot analysis of cultured endothelial cells derived from rat cerebral resistance vessels demonstrated the presence of c-mas mRNA. This is the first description of mas expression in non-neuronal cells. c-mas message was not detectable in cultured endothelial cells derived from other vascular beds, including rat aorta and mesenteric resistance vessels. Since c-mas has been purported to regulate the proliferation response to angiotensin, the growth properties of all three endothelial cell cultures were examined. The data indicated no differences in either basal or angiotensin-stimulated cell growth among brain, mesenteric or aortic-derived endothelial cells. These data suggest that c-mas is selectively expressed in brain endothelial cells and that this proto-oncogene does not regulate cell proliferation in this cell type.

Angiotensin II and atrial natriuretic factor receptor interactions at the blood-brain barrier.

Data from several laboratories indicate that cerebral endothelial cells possess cell surface receptors for numerous vasoactive agents including angiotensin II (AII) and atrial natriuretic factor (ANF). The intracellular messengers of these receptors as well as possible receptor interactions were explored. ANF increased cGMP 10-fold over basal levels while incubation of the microvessels with AII did not significantly affect the level of this nucleotide. In contrast, AII significantly potentiated the increase in cGMP by ANF. Incubation of cerebral microvessels with AII resulted in a significant increase in the intracellular mediator of PI hydrolysis, 1,2-diacylglycerol (DG). ANF had no affect on DG or on the AII mediated increase of DG. Finally, data at the level of receptor binding indicated that while ANF decreased [3H]angiotensin binding to cerebral microvessels, AII had no effect on the binding of ANF to its receptor. The results of the present study demonstrate that AII can potentiate the regulation of cGMP by ANF and suggest the possibility of receptor interactions in control of blood-brain barrier function.

Lysosomal enzymes in experimental diabetic cardiomyopathy.

The present study examines the role of cardiac lysosomal enzymes in the pathogenesis of the cardiomyopathy that develops in genetically diabetic C57BL/KsJ db+/db+ mice. Db+/db+ mice and littermate controls were sacrificed as age-matched pairs between 5-26 weeks of age. C57BL/6J ob/ob mice and littermates served as other controls. The hearts were excised, homogenized, and the following enzymatic activities measured: N-Acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-glucosaminidase, aryl sulphatase, alpha-mannosidase, alpha-glucosidase, beta-galactosidase, beta glucosidase, total p-nitrophenyl phosphatase, acid phosphatase and 5'-phosphodiesterase type IV. There is a progressive decrease in cardiac lysosomal enzyme activities of db+/db+ mice for the period 5-21 weeks of age. All enzyme activity is depressed significantly during the 9-21 week interval with beta-glucuronidase, aryl sulphatase and beta-glucosidase decreased about 40-50%. The decrease in lysosomal enzyme activity can explain the accumulation of large residual bodies and interstitial material in the myocardium of the db+/db+ animals

Lap Band adjustable gastric banding system: the Italian experience with 1863 patients operated on 6 years.

BACKGROUND: The Lap Band system procedure is currently the most common bariatric surgical procedure worldwide. This is an interim report of the experience of the 27 Italian centers participating in the national collaborative study group for Lap Band (GILB). METHODS: An electronic database was specifically created. It was mailed and e-mailed to all of the surgeons now performing the laparoscopic gastric banding operation in Italy. RESULTS: Beginning in January 1996, 1893 patients were recruited for the study. There were 1534 women and 359 men with a mean body mass index (BMI) of (range 30.4-83.6) and a mean age of 37.8 +/- 10.9 years (range; 17-74). The mortality rate has been 0.53% (n = 10), mainly due to cardiovascular complications (myocardial infarction, pulmonary embolism). The laparotomic conversion rate has been 3.1% (59/1893) and was higher in superobese patients (BMI>50) than in to morbidly obese patients (BMI <50) (p <0.05). Postoperative complications occurred in 193 patients (10.2%), including tube port failure (n = 79; 40.9%), gastric pouch dilation (GPD) (n = 93; 48.9%), and gastric erosion (n = 21, 10.8%). Most GPD (65.5%) occurred during the first 50 patients treated at each center. The incidence of GPD decreased as the surgeons acquired more experience. Surgery for complications was often performed by laparoscopic access, rarely via laparotomy. No death was recorded as a consequence of surgery to treat complications. Weight loss has been evaluated at the following intervals: 6, 12, 24, 36, 48, 60, and 72 months, with BMI 37.9, 33.7, 34.8, 34.1, 32.7, 34.8, and 32. CONCLUSIONS: The Lap Band system procedure has a very low mortality rate and a low morbidity rate and it yields satisfactory weight loss. Surgery for complications can be performed safely via laparoscopic access.

Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.

This report describes the initiation, cloning and establishment of long-term serial cultures of rat heart-derived vascular endothelial (EC) and smooth muscle cells (SMC). Populations of these cells derived from both the macro-and microcirculation were obtained utilizing isolated heart perfusion technique. Elimination of potential mesothelial cell contamination was achieved by ethanol fixation of the pericardial surface prior to perfusion. Initial outgrowths from perfusate yielded both endothelial (rapid adhering) and smooth muscle (slow adhering) appearing cell populations. Subsequent pooling of individual EC colonies resulted in maintaining, with gradual subcultivation, a stable homogeneous population which was designated RHE-parent. Upon continual subculture late passage (greater than P10) RHE-parent cell cultures expressed a marked heterogeneity in endothelial phenotypes. Cloning experiments resulted in establishing two distinct EC populations designated RHE-clone 1A and RHE-clone 2A. All RHE cell cultures exhibited the typical cobblestone growth pattern and positive immunofluorescent staining for factor VIII related antigen. In contrast, rat heart-derived smooth muscle cell (RH-SMC) cultures displayed the typical multilayered 'hill and valley' pattern and positive fluorescence for SMC-specific actin and myosin antibodies. Additional EC preparations, obtained without prior fixation of the pericardial surface, revealed cell clusters which stained positive for cytokeratin. On the other hand, RHE parent and cloned populations stained exclusively for vimentin, further confirming the absence of mesothelial cell contamination in these cultures. Cell growth studies on early (less than P10) and late (greater than P10) passage RHE-parent population revealed markedly different cell growth responses and cell morphology. Both EC cloned populations and more notably RHE-parent (less than P10) cultures were capable of significant growth when maintained in limiting serum concentration. Growth studies using serum-free RHE-parent conditioned medium demonstrated mitogenic activity when tested on RHE-parent cultures indicating the presence of an endothelial cell-derived growth factor. These studies indicate that long-term RHE and RH-SMC derived cell cultures can serve as a useful model to study the biology of vascular cells derived from different sites. In addition the demonstration of mitogenic activity in these cultures will enable us to explore further the nature of this response and compare this phenomenon with growth factors identified in large vessel cell systems.

Isolation and characterization of cerebral resistance vessel endothelium in culture.

Organ-derived endothelia have been shown to exhibit distinct patterns of morphology and growth responsiveness in vitro. This report describes the development, cloning and establishment of long-term serial cultures of rat vascular endothelial cells derived from cerebrocortical resistance vessels (small arteries and arterioles). Modification of our previous published technique for establishing resistance vessel-derived smooth muscle cells (RV-SMC) resulted in enhanced levels of endothelial outgrowth from collagenase-treated microvessel fragments. Although primary culture growth consisted predominantly of SMC, subsequent subcultivation of these cultures revealed the presence of distinct endothelial cell clusters within the SMC monolayer. Serial cloning of these isolates resulted in a homogeneous population of cells with the characteristic endothelial cobblestone growth pattern and positive immunofluorescence for factor VIII-related antigen. Previously established RV-SMC frozen stocks provided an additional source for obtaining resistance vessel endothelial cells. This was made possible by the slow proliferation rate of early-passage RV-SMC and their inability to withstand freezing procedures. Endothelial cells from both preparations were identical and designated resistance vessel derived endothelial cells RV-EC. Upon long-term cultivation (> P15), confluent RV-EC cultures expressed spontaneous multicellular cord development that stained positive for factor VIII-related antigen. Cell growth studies demonstrated that RV-EC were capable of significant growth when maintained in serum-free conditions. Growth kinetics using serum-free conditioned medium demonstrated mitogenic activity indicating the presence of an autocrine growth factor. Increase growth responsiveness was also noted in RV-EC when treated with a variety of peptide growth factors. These results indicate that resistance vessel endothelium can be successfully isolated and maintained in long-term serial cultures. Furthermore, the availability of cultured EC and SMC from this unique microvascular site will enable examination of cerebrovascular endothelial-smooth muscle cell interactions in vitro and may help to elucidate the mechanisms of altered vascular function in disease states.