Preparation and pharmaceutical evaluation for clinical application of high specific activity S-(-)[11C]CGP-12177, a radioligand for beta-adrenoreceptors.

BACKGROUND: Although S-(-)[C]CGP-12177 is a useful positron emission tomography (PET) ligand for beta-adrenoreceptors, the difficulty in radiolabelling the compound has prevented its extensive clinical application. Recently, we have developed a simple synthesis method for S-(-)[C]CGP-12177. In the present study, we attempted to prepare S-(-)[C]CGP-12177 with a high specific activity for intravenous injection which is feasible for the clinical evaluation of beta-adrenoreceptors. METHODS: The [C]methane produced during irradiation of a N2--H2 (95/5) mixture with an 18 MeV proton beam (20 microA, 30 min) was chlorinated using Cl2 to yield [C]carbon tetrachloride. S-(-)[C]CGP-12177 was synthesized by reacting the diamino precursor with [C]phosgene produced by oxidizing [C]carbon tetrachloride on a Fe--Fe2O3 column. The product was purified by using reversed phase, high-performance liquid chromatography (RP-HPLC) and the radioactive fraction containing S-(-)[C]CGP-12177 was collected and evaporated to dryness. S-(-)[C]CGP-12177 dissolved in physiological saline was sterilized through a 0.22 microm membrane filter. The radiochemical purity and the mass of the compound were determined with RP-HPLC. The residual organic solvents were determined with GC. Tests for sterility and the presence of bacterial endotoxins were also performed. RESULTS: S-(-)[C]CGP-12177 for intravenous injection was prepared in 25 min after the end of bombardment with a yield of 1.5+/-0.2 GBq. Specific activity was found to be 385.4+/-133.0 GBq/ micromol at the end of synthesis (EOS) (n=3). Radiochemical purity was found to be more than 99%. Toluene was not detected in the solution. The ethanol concentration was determined to be 60.3+/-52.5 ppm. Tests for sterility and bacterial endotoxins showed negative results. CONCLUSION: We successfully prepared S-(-)[C]CGP-12177 formulated for intravenous injection with high purity and high specific activity, which is feasible for the clinical evaluation of beta-adrenoreceptors.

Expression and properties of human liver beta-ureidopropionase.

A cDNA encoding beta-ureidopropionase (BUP) was isolated from a human liver cDNA library, expressed in E. coli, and purified from the culture extract. The 2,006 bp cDNA contained a 1,152 bp open reading frame encoding a protein of 384 amino acids with a molecular weight of 43,165 Da. The subunit molecular weight of the enzyme expressed was about 43,000 Da. The enzyme was inhibited by 1 mM propionate, but not by 10 mM beta-alanine. Chemical analysis of the purified human BUP showed 0.54 zinc atoms per subunit, and the sequence of BUP cDNA contained one putative zinc-binding site motif. The purified enzyme had a pI of 5.65, and exhibited positive cooperativity with N-carbamoyl-beta-alanine as the substrate with a Hill coefficient 2.0. These properties of human BUP, except the inhibition by beta-alanine, were similar to the rat liver purified enzyme. Beta-alanine inhibits rats BUP activity. The complex regulatory function and the negative cooperative mechanism of BUP by beta-alanine have been observed in rats. This kind of mechanism may not exist in humans, because beta-alanine did not inhibit human BUP.

Abnormal neuronal network in anorexia nervosa studied with I-123-IMP SPECT.

Single photon emission computed tomography was used to study 14 female patients with anorexia nervosa and 8 female normal comparison subjects. Automatic voxel-based analysis of the images was carried out using statistical parametric mapping (SPM) software. Statistics across the entire brain were displayed as Z scores (threshold: P<0.05). Compared with the normal comparison subjects, the anorectic patients were characterized by hypoperfusion in the medial prefrontal cortex and the anterior cingulate gyrus, and hyperperfusion in the thalamus and the amygdala-hippocampus complex. These results suggest that a dysfunction in neuronal circuitry may be related to anorexia nervosa.

Comparison of (18)F-FDG, (131)I-Na, and (201)Tl in diagnosis of recurrent or metastatic thyroid carcinoma.

UNLABELLED: There are several reports about the usefulness of (18)F-FDG PET in thyroid cancer. However, few studies have compared FDG PET with (131)I and (201)Tl scintigraphy. The aim of this study was to evaluate the clinical significance of whole-body FDG PET in differentiated thyroid cancer and to compare the results with those obtained from (131)I and (201)Tl scintigraphy. METHODS: Whole-body FDG PET was performed on 32 patients (10 men, 22 women; age range, 30-77 y; mean age, 54 y) with differentiated thyroid cancer (5 cases of follicular cancer and 27 of papillary cancer) after total thyroidectomy. An overall clinical evaluation was performed, including cytology, thyroglobulin level, sonography, MRI, and CT, to allow a comparison with functional imaging results for each patient. Metastatic regions were divided into five areas: neck, lung, mediastinum, bone, and other. Multiple lesions in one area were defined as one lesion. The tumor-to-background ratio (TBR) was measured for the lesions that were positive for both (201)Tl uptake and FDG PET uptake. RESULTS: The number of lesions totaled 47. Forty-one (87%) were detected by all scintigraphic methods. FDG uptake was concordant with (131)I uptake in only 18 lesions (38%). FDG uptake was concordant with (201)Tl uptake in 44 lesions (94%). Only one lesion was negative for FDG uptake and positive for (201)Tl uptake, and two lesions were positive for FDG uptake and negative for (201)Tl uptake. A significant correlation was seen between the TBR of (201)Tl and that of FDG (r = 0.69; P<0.05). CONCLUSION: These data indicate that for detecting metastatic lesions, FDG PET and (131)I scintigraphy may provide complementary information, whereas FDG PET may provide results similar to those of (201)Tl scintigraphy. Thus, the combination of (131)I scintigraphy and FDG PET (or (201)Tl scintigraphy) is the method of choice for detecting metastatic thyroid cancer after total thyroidectomy.

Thalamic asymmetry on interictal SPECT in patients with frontal lobe epilepsy.

BACKGROUND: Interictal brain single photon emission computed tomography (SPECT) is useful for the detection of seizure focus. Recent reports indicate a hypoperfusion in the ipsilateral thalamus as a seizure focus on interictal SPECT in temporal lobe epilepsy. In frontal lobe epilepsy (FLE), however, the alteration of perfusion in the thalamus has not been well documented. This study aimed to assess whether perfusion analysis on the thalamus may add useful information for the detection of epileptic foci in patients with FLE. METHODS: Interictal brain SPECT was performed in 11 patients with FLE. The asymmetry index for the thalamus and frontal area in the SPECT image was calculated in order to compare the laterality of the seizure foci. RESULTS: Thalamic asymmetry was seen in seven patients (64%), while cortial asymmetry was seen in six patients (55%). The concordance with the lateralization of the seizure foci was 6/7 (86%) in the thalamus, and 4/6 (67%) in the frontal area. Four patients showed only thalamic asymmetry. Concordance with the lateralization of the seizure focus was found in all of them. CONCLUSION: These preliminary results suggest that hypoperfusion in the thalamus may have a complementary role to lateralize the epileptic foci in patients with FLE.

Serial changes in cerebral blood flow and flow-metabolism uncoupling in primates with acute thromboembolic stroke.

The authors recently developed a primate thromboembolic stroke model. To characterize the primate model, the authors determined serial changes in cerebral blood flow (CBF) and the relation between CBF and cerebral metabolic rate of glucose (CMRglc) using high-resolution positron emission tomography. Thromboembolic stroke was produced in male cynomolgus monkeys (n = 4). Acute obstruction of the left middle cerebral artery was achieved by injecting an autologous blood clot into the left internal carotid artery. Cerebral blood flow was measured with [15O]H2O before and 1, 2, 4, 6, and 24 hours after embolization. CMRglc was measured with 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) 24 hours after embolization. Lesion size and location 24 hours after embolization was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) staining method. The results are summarized as follows: (1) 1 hour after embolization, CBF in the temporal cortex and the basal ganglia decreased to < 40% of the contralateral values. In these regions, regarded as an ischemic core, CBF decreased further with time and CMRglc at 24 hours also decreased. Infarcted lesions as indicated by being unstained with TTC were consistently observed in these regions. (2) In the parietal cortex and several regions surrounding the ischemic core, CBF was > 40% of the contralateral values 1 hour after embolization and recovered gradually with time (ischemic penumbra). In these regions, CMRglc at 24 hours increased compared with that in the contralateral regions, indicating an uncoupling of CBF and CMRglc. No obvious TTC-unstained lesions were detected in these regions. The authors demonstrated a gradual recovery of reduced CBF, an elevated CMRglc and a CBF-CMRglc uncoupling in the penumbra regions of the primate model. Positron emission tomography investigations using this model will provide better understanding of the pathophysiology of thromboembolic stroke in humans.

cDNA cloning of an alternative splicing variant of protein kinase C delta (PKC deltaIII), a new truncated form of PKCdelta, in rats.

Recently, an alternative splicing variant of mouse protein kinase C delta (PKC deltaII, GenBank Accession No. AB011812) has been reported which has a 78 bp (26 amino acid) insertion at the caspase-3 recognition sequence in the V3 region of PKC delta (PKC deltaI). We isolated a cDNA encoding a new variant of PKC delta (PKC deltaIII, AF219629), which has a 83 bp insertion at the same site in the V3 region, by RT-PCR using rat testis RNA as a template. In rats, the 83 bp insertion causes inframe termination, and rat PKC deltaIII protein is expressed as a truncated form, having only the regulatory domain without a catalytic domain. Genomic DNA analysis revealed that the difference between mouse PKC deltaII and rat PKC deltaIII is derived from the different sequence at the 5'-splicing donor sites. To investigate the potential functions of the truncated form of PKC delta, rat PKC deltaIII fused to green fluorescent protein (GFP) was expressed in CHO-K1 cells. PKC deltaIII-GFP was localized in the cytoplasm with dot-like accumulation and highly expressed on the plasma membrane, whereas PKC deltaI-GFP is localized homogeneously throughout the cytoplasm, including the nucleoplasm. Stimulation by phorbol ester caused weak translocation of deltaIII-GFP from the cytosol to the plasma membrane. These results suggest that PKC deltaIII may show a dominant negative effect against PKC deltaI, and that the modulation of signal transduction by alternative splicing variant may play a crucial role in the physiological and/or pathological conditions, and the pathogenesis of disease.

Ischemic "cross" tolerance in hypoxic ischemia of immature rat brain.

The phenomenon of ischemic tolerance has been closely associated with the expression of heat shock proteins but recently, stress tolerance not related to hsp72 has been reported. In the present study, we focused on ischemic tolerance induced by hypoxia and hyperthermia in neonatal rat brain and analyzed the expression of hsp72. In a neonatal rat model of hypoxic ischemia (H-I), preconditioning by whole-body hyperthermia or hypoxia was induced 24 h prior to the ischemia. Brain damage was histologically evaluated and the expressions of hsp72 were analyzed. Hyperthermic preconditioning at 41 degrees C for 15 min, as well as hypoxic preconditioning with 8% hypoxia for 3 h, had almost complete neuroprotective effects. However, we failed to detect the expression of hsp72 in any of preconditioning. Only the H-I insult itself induced hsp72 in the dorsal striatum and slightly in the thalamus and the hippocampus. Hyperthermic preconditioning has neuroprotective effects which are comparable to hypoxic preconditioning in immature brain. The expression of hsp72 is not likely necessary for the ischemic tolerance in immature brain.

The correlation between feed-intake cycle and nutritional zinc-deficient status in rats.

The characteristic cyclic variation in feed intake of rats fed a Zn-deficient diet (Mills et al, Am J Clin Nutr 22: 1240-1249 (1969)) followed a Cosinor curve, as determined by computer analysis (Tamaki et al, Br J Nutr 73: 711-722 (1995)). The values of amplitude for the feed-intake cycle had a positive correlation to their own day-to-day variations and to the correlation value of their own simulated cycles (r2 = 0.764, df = 50, p < 0.001 and r2 = 0.682, df = 50, p < 0.001, respectively). The cyclic variation in feed intake was accompanied by a cyclic variation in body-weight change in rats fed the Zn-deficient diet, and cyclic variation in body-weight change occurred similarly in pair-fed control rats. There were no differences in the mesors of body-weight change cycles of Zn-deficient rats and pair-fed control rats (Zn-deficient rats: 2.5 +/- 1.0 g/d, pair-fed rats: 2.8 +/- 1.0 g/d, mean +/- SD, df = 18, t = -0.674, ND). Rats fed the Zn-deficient diet were given different amounts of Zn supplementation by daily subcutaneous injection. The amplitude of the feed-intake cycle was decreased with increasing Zn supplementation (r2 = 0.919, df = 5, p < 0.001). The concentration of Zn for the appearance of the feed-intake cycle was estimated to be 71.6 +/- 6.6 micrograms/d per rat. The Zn level in the serum showed a significant decrease in the Zn-deficient diet groups, but the supplement of Zn did not vary in the Zn-deficient rats injected with up to 47.3 micrograms/d per rat. From these results, an analysis of the feed-intake cycle allowed us to estimate the quantitative Zn-deficient status of rats.

Cosinor analysis of feed intake cycle of rats fed a zinc-deficient diet and the effect of zinc supplementation.

Rats fed a Zn-deficient diet develop a characteristic cyclic variation in feed intake (Mills et al., Am J Clin Nutr 22: 1240-1249 (1969). A preliminary analysis (Tamaki et al., Br J Nutr 73: 711-722 (1995)) of the cyclic variations was followed with a personal computer. Cosinor analysis revealed that the cyclic period of the feed intake of male rats was 3.5 +/- 0.05 d. The mesor, amplitude and acrophase value were 10.0 +/- 0.3 g/d, 4.4 +/- 0.2 g/d and 3.5 +/- 0.3 radian, respectively. The cycle of body-weight change of the Zn-deficient rats was well synchronized with that of feed intake. The parameters of the feed intake cycle had a high correlation to the corresponding parameters of body-weight change (mesor: r = 0.846; amplitude: r = 0.771; period: r = 0.925; acrophase: r = 0.452). With the supplementation of Zn (0.95-3.80 mg/kg of the Zn-deficient diet), cyclic variations in feed intake and body-weight change were also found. The mesor, amplitude and period of feed intake cycle were is good correlation with Zn intake (r = 0.856, p < 0.001, r = 0.804, p < 0.001 and r = 0.613, p < 0.01, respectively). The cycle of feed intake of the rats fed a Zn-free diet was simulated to be: mesor 9.7 +/- 0.1 g/d, amplitude 6.5 +/- 0.1 g/d and period 3.4 +/- 0.02 d. The concentration of Zn intake given the half-maximal value of the amplitude was assumed to be 56 +/- 1 microgram/d.

Thrombolytic therapy with tissue plasminogen activator for prevention of vasospasm in experimental subarachnoid hemorrhage: its efficacy and problems.

We investigated the efficacy of two different tissue plasminogen activators (t-PA) for preventing vasospasm after experimental subarachnoid hemorrhage (SAH) in rabbits. Intrathecal injection of Silteplase and Alteplase showed significant preventive action against vasospasm following SAH and thrombolytic effect. The low dose groups with both t-PA showed more preventive action on day 1 than the high dose groups. The data suggest that the determination of optimum dose of t-PA is essential in clinical use of t-PA.

[Currarino triad : a case report].

The authors report a case of Currarino triad comprising anorectal malformations, sacral bony anomaly and presacral mass. A 1-year-old boy was presented with constipation as his chief complaint. No neurological deficit was found on admission. There was no cutaneous evidence of underlying spinal dysraphism. Lumbar X-ray films showed bony defect caudal to the third sacral vertebra. A barium-enema examination revealed an anterior displacement of the rectum. A myelography showed a presacral cavity filled with contrast medium. MRI demonstrated a thick filum terminale, and a round hypointense mass in the pelvis on T1 weighted images and hyperintense on T2 weighted images. Surgically we released the thick filum terminale, and obliterated the anterior sacral meningocele, because total removal would have been hazardous. Postoperatively transient dysuria was observed for a month, and the difficulty in defecation persisted. Recognition of this rare condition will lead to correct diagnosis and proper treatment.

Value of fluorine-18-FDG-PET to monitor hepatocellular carcinoma after interventional therapy.

METHODS: Thirty-two tumors in 30 patients with hepatocellular carcinoma (HCC) were studied preoperatively using PET with 18F-labeled 2-fluoro-2-deoxy-D-glucose (FDG) to evaluate the metabolic activity of the lesions after interventional therapy. All patients had received transcatheter arterial chemoembolization therapy using iodized oil (Lipiodol, Laboratoire Guerbet, Alnaysous-Bois, France) before the PET study. The tumors were 2 to 18 cm in diameter. FDG uptake at 48 to 60 min after tracer injection was used to determine the standardized uptake value (SUV). The SUVs of the tumor and nontumor regions of the liver were calculated to obtain the tumor-to-nontumor ratio (SUV ratio). The PET results were compared with the findings of CT and histologic examination. RESULTS: The tumors were divided into three types, consisting of those with increased FDG uptake (SUV ratio of 1.07-2.66, Type A, n = 19), similar FDG uptake to the surrounding nontumor region (SUV ratio of 0.77-1.04, Type B, n = 7) and decreased or absent FDG uptake (SUV ratio of 0.13-0.58, Type C, n = 6). In histologic examination, viable HCC tissue remained in all Type A and B tumors, whereas more than 90% necrosis was found in the Type C tumors, indicating that interventional therapy had been effective. These PET findings reflected tumor viability more accurately than the extent of intratumor Lipiodol retention on CT images. CONCLUSION: FDG-PET appears to be a valuable method for the assessment of tumor viability after interventional therapy for HCC.

Clinical evaluation of cardiac function by ambulatory ventricular scintigraphic monitoring (VEST): validation and study of the effects of nitroglycerin and nifedipine in patients with and without coronary artery disease.

Global left ventricular function and ECGs were continuously monitored by radionuclide ambulatory ventricular function monitoring (VEST) and validated against multigated blood pool analysis (MUGA) and left ventriculography in 26 subjects (study 1). Ejection fraction by VEST (Y) showed good correlation with Y = 5.5 +/- 0.79 X (r = 0.91), Y = 1.7 +/- 0.86 X' (r = 0.91), and Y = 11.6 + 0.68 X" (r = 0.82) to sitting and supine MUGA and left ventriculography, respectively. In study 2 left ventricular function and ECGs were evaluated at rest and during exercise without any drug (control), with nitroglycerin, and with nifedipine in 21 patients with coronary disease (group I) and six normal subjects (group II). In group I abnormal ejection fraction responses (exercise increase less than or equal to 6%) during the control exercise period were found in 15 patients (71%), ST segment abnormalities in seven (33%), and chest pain in four (18%). Control exercise increased end-diastolic volume (100 to 112 +/- 8%) and end-systolic volume (53 +/- 15% to 63 +/- 22%) and decreased the ejection fraction (47 +/- 15% to 43 +/- 21%). The ejection fraction during exercise increased after nitroglycerin (50 +/- 22%) or nifedipine (54 +/- 21%) (p less than 0.05). In group II the ejection fraction was unchanged between rest and exercise with or without nitroglycerin or nifedipine. Thus combined radionuclide and ECG monitoring by VEST could detect changes in left ventricular function at rest and during exercise over a prolonged period and demonstrated that nitroglycerin and nifedipine improved cardiac function in the ischemic setting with an increased ejection fraction in the upright position.

Cerebral energy metabolism in experimental canine hydrocephalus.

Cerebral energy metabolism, its relationship to the stage and extent of hydrocephalus, and the effect of cerebrospinal fluid (CSF) removal on it were studied in experimental canine hydrocephalus produced by intracisternal injection of kaolin by using phosphorus-31 (P-31) magnetic resonance (MR) spectroscopy and MR imaging. P-31 MR spectra were serially obtained before and after CSF removal, maximally on eight occasions over a period of nearly 5 h. There was a decrease in the ratio of creatine phosphate to inorganic phosphate, used as an indicator of the bioenergetic state, in acute and subacute stages of hydrocephalus as compared with the control. An animal in the subacute stage, when periventricular edema was most prominent, exhibited the most predominent decrease in this ratio at 14 days after hydrocephalic insult. The recovery of the ratio toward the control level was seen in the chronically hydrocephalic animal. There was no change in adenosine triphosphate (ATP) levels in any stage of hydrocephalus. Serial spectra obtained after the withdrawal of ventricular CSF showed no change in the bioenergetic state of the brain in any stage of hydrocephalus. There was no relationship between either the extent of hydrocephalus or the ventricular CSF pressure and the change in the bioenergetic state or the levels of any of the phosphorus compounds. These findings may indicate the alteration of the mitochondrial energy metabolism in hydrocephalus, which may explain the mechanism of hydrocephalic syndrome.

Germ cell tumors of the thalamus and the basal ganglia.

Two cases of germ cell tumors (GCTs) of the basal ganglia are presented and 40 previously reported cases are reviewed. The incidence of GCTs of the basal ganglia and thalamus was estimated as less than 14% of all intracranial GCTs. All patients except for two (95%) were male, aged 7-19 years. The clinical course was usually slow. The major symptoms were hemiparesis, mental deterioration such as dementia or character change, precocious puberty, diabetes insipidus, oculomotor palsy, speech disturbance, and hemianopsia. Signs of intracranial hypertension did not occur until the late stages of the disease. The plain CT finding was characterized by an irregularly defined, slightly high-density area frequently accompanied by central low-density areas without significant mass effect. The tumors showed mild to moderate and nonhomogeneous contrast enhancement. An ipsilateral cerebral hemiatrophy was often found. MR images demonstrated the corresponding findings. GCTs of the basal ganglia had a high possibility of containing components other than germinoma such as choriocarcinoma, endodermal sinus tumor, and embryonal carcinoma. Thus, tumor markers in the serum, CSF, or cyst fluid were frequently positive. With recent refinement of microsurgical techniques as well as immunohistochemical study and measurements of tumor markers of serum, CSF, and cyst fluid, major resections of tumor, accurate pretreatment histologic diagnosis, and early determination of the specific types of this tumor appear to be readily possible. This is essential for effective treatment of patients not only with radiosensitive germinoma, but also those with radioinsensitive nongerminoma variants and a combination of them located in this region.

Metastatic nodules of hepatocellular carcinoma: detection with angiography, CT, and US.

Accurate detection of intrahepatic metastases, or daughter nodules, of primary hepatocellular carcinoma is of crucial importance. Due to the introduction of infusion hepatic angiography, computed tomography (CT) after Lipiodol (iodized oil) infusion, and intraoperative ultrasound (US), tumors less than 10 mm in diameter are now frequently found. We compared the diagnostic accuracy of these three modalities in the detection of nodules in 45 patients who had hepatocellular carcinoma (confirmed by biopsy). CT with Lipiodol was superior to hepatic angiography in demonstrating nodules when they were overlapped by the primary tumor or very small in size. Intraoperative US demonstrated nodules in four avascular or hypovascular hepatocellular carcinomas, which both hepatic angiography and CT failed to demonstrate. In cases associated with severe liver cirrhosis, differentiation of small nodules from regenerating cirrhotic nodules was sometimes difficult with intraoperative US. The combined use of these three modalities is indispensable for the accurate detection of small nodules of metastatic hepatocellular carcinoma.

[Intracranial anomalies in myelomeningocele--observation with magnetic resonance (MR) imaging].

It is generally accepted that myelomeningocele frequently associates with Arnold-Chiari malformation and other anomalies of the intracranial structures. The ventriculographic and CT findings of the patients with myelomeningocele has been reported. Magnetic resonance (MR) imaging is useful to observe the coronal and sagittal images of the brain in order to speculate the etiological mechanism of myelomeningocele and its associated anomalies. We experienced three cases of myelomeningocele and reviewed their MR images using coronal and sagittal tomography in spin echo and inversion recovery technique. The morphological detail of MR images as to the intracranial structures was presented. Possible mechanism of the anomalous structures of the brain in myelomeningocele was also described.