Transport of amino acid amide sarcosinamide and sarcosinamide chloroethylnitrosourea in human glioma SK-MG-1 cells.

The transport of the amino acid amide N-[3H]sarcosinamide (methyl glycinamide) was investigated in human glioma SK-MG-1 cells. Sarcosinamide uptake was found to be temperature dependent, sodium independent, and linear up to 1 min at 22 degrees C. Equilibrium was reached after 10 min at 22 degrees C with accumulation slightly above unity. Sarcosinamide was not metabolized in the cells as shown by thin layer chromatography. The uptake of sarcosinamide was significantly decreased when the extracellular pH was lowered from 7.5 to 6.0 and significantly enhanced at pH values above 7.5. The latter effect may be due mainly to increased cell permeability at high pH. The uptake of the labeled sarcosinamide was trans-stimulated by excess cold sarcosinamide. Sarcosinamide uptake over a 200-fold range of concentrations followed Michaelis-Menten kinetics with a Km of 0.284 +/- 0.041 mM and a Vmax of 0.154 +/- 0.024 nmol/10(6) cells/min. The uptake of sarcosinamide was significantly reduced by iodoacetate but not by the metabolic poisons NaF, ouabain, or dinitrophenyl, suggesting that the uptake is not dependent on energy, rather it proceeds by facilitated diffusion. Several naturally occurring substrates were unable to inhibit the uptake of sarcosinamide. Leucine significantly reduced the uptake of sarcosinamide, while sarcosinamide was a weak inhibitor of leucine transport. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid a specific substrate for the sodium-independent, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid-sensitive amino acid system L failed to inhibit the uptake of sarcosinamide. Epinephrine reduced the uptake of sarcosinamide and sarcosinamide was equally potent as an inhibitor of epinephrine transport. Dixon plot analysis demonstrated that epinephrine (Km = 0.270 mM) inhibits the uptake of sarcosinamide competitively (Ki = 0.260 mM). These results indicate that sarcosinamide is a substrate for the catecholamine transporter. The alkylating agent, sarcosinamide chloroethylnitrosourea, was tested for its ability to inhibit the uptake of sarcosinamide. The results of Dixon plot analysis were consistent with competitive inhibition of sarcosinamide uptake and the inhibition constant Ki for SarCNU was found to be 3.26 +/- 0.57 mM. The steady-state intracellular concentration of SarCNU was found to be significantly higher (cell:medium ratio of 1.03 +/- 0.01) than that of BCNU cell:medium ratio of 0.52 +/- 0.12). These findings indicate that SarCNU and sarcosinamide share the same carrier for uptake in SK-MG-1 cells. This transport mechanism may be responsible for the increased accumulation of SarCNU as compared to BCNU, a nitrosourea which enters cells by passive diffusion.

Pharmacokinetics of positron-labeled 1,3-bis(2-chloroethyl)nitrosourea in human brain tumors using positron emission tomography.

The nitrosoureas are widely used in the chemotherapy of brain tumors, two of the most common being 1,3-bis(2-chloroethyl)nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. However, we do not understand how these compounds work, nor do we know which part of the molecule has antitumor activity. In six patients with brain tumor, we measured the kinetic behavior of positron-labeled 1,3-bis(2-chloroethyl)nitrosourea in both the tumor and the normal brain with the aid of positron emission tomography; we also analyzed the distribution of radioactivity in plasma. We found the clearance of total radioactivity from the tumor to be significantly slower than from the contralateral brain and plasma, indicating a different rate of 1,3-bis(2-chloroethyl)nitrosourea decomposition in the tumor than in normal brain.

Evaluation of [18F]-4-fluoroantipyrine as a new blood flow tracer for multiradionuclide autoradiography.

This article reports the evaluation of [18F]-4- fluoroantipyrine (FAP) as a quantitative blood flow tracer by comparing blood flow measured with [18F]FAP to that determined simultaneously with [14C]-4-iodoantipyrine (IAP), a standard blood flow tracer, by means of double-tracer autoradiography. The single-pass extraction value (m), which indicates diffusibility of a tracer, was determined according to the procedure described by Crone . The diffusibility of FAP was essentially the same as that of IAP. The brain-blood partition coefficient for FAP was found to be similar to that for IAP, 0.89 +/- 0.01. Values of local cerebral blood flow obtained with FAP agree with those determined with IAP. From these results, we concluded that FAP is indeed as good a blood flow tracer as IAP. Since 18F is a positron-emitting radionuclide, it might be a useful tracer for blood flow measurement by positron emission tomography.

Quantification of glucose utilization in an experimental brain tumor model by the deoxyglucose method.

Reevaluation of lumped and rate constants is necessary when Sokoloff's 2-deoxyglucose (DG) method is used to measure glucose utilization in pathological tissue. We describe here a modification of Sokoloff's lumped constant measurement that permits simultaneous estimation of both lumped and rate constants from a single animal experiment. A subcutaneous tumor model (AA ascites tumor) was used for measurement of these constants with a procedure similar to Sokoloff's that kept the plasma tracer concentration constant. Measured constants were as follows: lumped constant, 0.654 +/- 0.081; k1, 0.196 +/- 0.038 min-1; k2, 0.262 +/- 0.067 min-1; k3, 0.117 +/- 0.044 min-1. These constants were used to quantify glucose utilization in the implanted brain tumor. To test the validity of this method, we compared a fraction of the free DG pool calculated using the tumor constants with a fraction measured directly by chromatographic analysis of tissue samples from both subcutaneous tumor and implanted brain tumor. The values derived by chemical analysis agreed well with those predicted by the calculations. The value of k4 varied from 0.0031 +/- 0.0018 min-1 for the tumor tissue to 0.0214 +/- 0.0024 min-1 for tumors with a large necrotic center. This method would be especially useful when applied to xenograft human gliomas in nude mice for quantification of glucose utilization in human gliomas by means of positron emission tomography.

An improved approach for measurement of regional cerebral rate constants in the deoxyglucose method with positron emission tomography.

A new experimental approach was designed to measure regional rate constants for [18F]2-fluoro-2-D-deoxyglucose in the three-compartment model. A programmed infusion was used to keep the plasma tracer concentration constant for the first 20 min. Positron emission tomography images of the brain were taken every minute for 20 min in a low-resolution mode, then every 15-20 min for 2-3 h in a medium-resolution mode. Two simplified operational equations for the calculation of the regional rate constants were derived that incorporated the contribution of the vascular compartment to tissue activity. The first equation was applied to data collected during the initial 20 min (when the concentration of plasma tracer was constant) to estimate the values of k1*, k2*, k3*, and the fraction of the vascular compartment. The second equation was applied to data collected during the whole experimental period to find the value of k4* and to provide a better estimate of k3*. The regional rate constants measured experimentally in three dogs and a brain tumor patient agreed well with those in the literature. This method permits estimation of the local CMRglu under pathological conditions using regionally measured rate constants and provides new information on the pathophysiological meaning of the rate constants.

Entorhinal cortex in temporal lobe epilepsy: a quantitative MRI study.

BACKGROUND: The entorhinal cortex (EC) is a distinct anatomic and functional region of the anterior parahippocampal gyrus, which plays a role in seizure generation and propagation in temporal lobe epilepsy (TLE). In tissue resected from TLE patients, cell loss in the EC has been described. OBJECTIVES: To develop a standardized protocol for identifying the anatomic boundaries of the EC using high-resolution MRI and to examine morphologic changes of the EC in TLE. METHODS: We performed T1-weighted MRIs in 20 patients (7 males) with TLE (mean age 34 years) and 18 normal controls (mean age 26 years). Eleven patients had a left and 9 a right epileptic focus as defined by history, video-EEG, and surgical outcome. The volumes of the EC, the hippocampus, and the amygdala were measured using a standardized MRI protocol. Analysis of variance (ANOVA) was used to examine the effect of seizure focus lateralization and hemisphere on these volumes. An asymmetry ratio [A (%) = 100 x (R-L)/(R+L)/2] was also compared between groups using ANOVA. RESULTS: In normal controls the volume of the right EC was 1,247 +/- 127 mm3 (mean +/- standard deviation), and that of the left EC was 1,215 +/- 135 mm3 (p > 0.05). We found a bilateral reduction in the volume of the EC in TLE patients compared with controls (p < 0.05). Examination of the asymmetry ratios showed that the reduction in volume of the EC was greater ipsilateral to the epileptic focus (p < 0.05). The volumes of the hippocampus and the amygdala were smaller on the side of the focus in TLE patients compared with controls (p < 0.05). CONCLUSIONS: With a standardized protocol for the quantitative assessment of the EC, patients with unilateral TLE show bilateral reduction in the volume of the EC. However, this reduction is more severe ipsilateral to the epileptic focus.

Excitatory amino acids are elevated in human epileptic cerebral cortex.

We used intraoperative electrocorticography to identify and compare specimens from two groups of patients undergoing temporal lobectomy: (1) spiking cortex (12 patients)--epileptic activity recorded over much of the temporal convexity; and (2) nonspiking cortex (9 patients)--temporal convexity free of interictal spiking, epileptic activity confined to the hippocampus and/or amygdala. Comparative amino acid levels were (mumol/g protein, mean +/- SEM): glutamate--spiking 109.8 +/- 1.8, nonspiking 87.4 +/- 2.0 (p less than 0.001); aspartate--spiking 15.2 +/- 0.9, nonspiking 12.2 +/- 0.5 (p less than 0.05); GABA--spiking 15.0 +/- 1.0, nonspiking 13.9 +/- 1.4 (NS); taurine--spiking 14.5 +/- 0.8, nonspiking 12.2 +/- 0.8 (NS); and glycine--spiking 11.5 +/- 0.8, nonspiking 7.4 +/- 0.6 (p less than 0.01). Cortical epileptic activity appears to be associated with elevated concentrations of glutamate, aspartate, and glycine, but not GABA and taurine, perhaps indicating a relative imbalance between putative excitatory and inhibitory amino acid neurotransmitters.

Gelastic seizures and hypothalamic hamartomas: evaluation of patients undergoing chronic intracranial EEG monitoring and outcome of surgical treatment.

We retrospectively studied 12 consecutive patients with gelastic seizures and hypothalamic hamartomas who, because of intractable epilepsy, underwent chronic intracranial EEG monitoring or epilepsy surgery. All patients had medically refractory seizures that included laughter as an ictal behavior (gelastic seizures). The hypothalamic hamartomas were identified with neuroimaging studies (12 of 12) and by pathologic verification (four of 12). Associated clinical features included behavioral disorders (n = 5), developmental delay (n = 4), and precocious puberty (n = 2). Interictal extracranial EEG predominantly showed bi-hemispheric epileptiform changes suggesting a secondary generalized epileptic disorder. Intracranial EEG recordings, performed in eight patients, indicated the apparent focal onset of seizure activity (anterior temporal lobe [n = 7] and frontal lobe [n = 1]). None of the seven patients who underwent a focal cortical resection, however, experienced a significant reduction in seizure tendency. An anterior corpus callosotomy, performed in two patients with symptomatic generalized epilepsy, resulted in a worthwhile reduction in drop attacks. Results of this study may modify the surgical strategies in patients with gelastic seizures and hypothalamic hamartomas.

The role of auditory cortex in retention of rhythmic patterns as studied in patients with temporal lobe removals including Heschl's gyrus.

This experiment examined the participation of the auditory cortex of the temporal lobe in the perception and retention of rhythmic patterns. Four patient groups were tested on a paradigm contrasting reproduction of auditory and visual rhythms: those with right or left anterior temporal lobe removals which included Heschl's gyrus (HG), the region of primary auditory cortex (RT-A and LT-A); and patients with right or left anterior temporal lobe removals which did not include HG (RT-a and LT-a). Estimation of lesion extent in HG using an MRI-based probabilistic map indicated that, in the majority of subjects, the lesion was confined to the anterior secondary auditory cortex located on the anterior-lateral extent of HG. On the rhythm reproduction task, RT-A patients were impaired in retention of auditory but not visual rhythms, particularly when accurate reproduction of stimulus durations was required. In contrast, LT-A patients as well as both RT-a and LT-a patients were relatively unimpaired on this task. None of the patient groups was impaired in the ability to make an adequate motor response. Further, they were unimpaired when using a dichotomous response mode, indicating that they were able to adequately differentiate the stimulus durations and, when given an alternative method of encoding, to retain them. Taken together, these results point to a specific role for the right anterior secondary auditory cortex in the retention of a precise analogue representation of auditory tonal patterns.

An on-line synthesis of [15O]N2O: new blood-flow tracer for PET imaging.

This paper describes a recently developed on-line synthesis of a new blood-flow tracer, O-15-labeled nitrous oxide. The tracer was produced by catalytic oxidation of anhydrous ammonia in a gas mixture containing O-15-labeled molecular oxygen. (The oxygen-15 was produced by a 14N(d,n)15O reaction.) Anhydrous ammonia was mixed with the gas containing [15O]O2, and after preheating to about 200 degrees C was carried through an oven containing a Pt catalyst kept at about 310 degrees C. Labeled gas was purified in H3PO4 and KOH traps. O-15-labeled nitrous oxide was identified by gas radiochromatography and by various chemical reactions. Radiochemical purity of the O-15-labeled nitrous oxide exceeded 98%, radiochemical yield corrected for radioactioactive decay was 15-20%, and specific activity at the end of synthesis was about 50 mCi/mmole.

Radiation dose to upper airways from inhaled oxygen-15 carbon dioxide.

According to Powell et al., significant retention of 15O in the tracheal mucosa results in a radiation absorbed dose of 75 to 200 rad upon breathing of 15O-CO2 for one hour at 1 mCi/liter. Such a high dose would seriously compromise the 15O-CO2 inhalation method for positron emission tomographic (PET) measurement of cerebral blood flow (CBF). In order to verify these results, we have assayed 15O activity in the tracheal region of three volunteers by PET during inhalation of 15O-CO2 and 15O-O2. Using methods similar to those of the above authors for estimating absorbed dose in the tracheal mucosa, we have obtained a value of 14-38 rad, which is more in keeping with 3-5 rad found by Bigler et al. from direct assay of mucus and saliva. We conclude that the 15O-CO2 inhalation method is a safe and practical means of measuring CBF by PET.

Metabolic and hemodynamic evaluation of gliomas using positron emission tomography.

Positron emission tomography (PET) was used on 16 patients with untreated cerebral gliomas to measure cerebral glucose and oxygen metabolism, oxygen extraction, blood flow, and blood volume. In addition, pH values were obtained for seven cases. Gliomas were later proven by biopsy; two patients had tumors with degrees of malignancy of grade II, two patients had grade III, and 12 patients had grade IV tumors. Compared with homologous gray matter regions in the opposite hemisphere, tumor tissue showed increased blood volume, but decreased oxygen extraction and oxygen metabolism. Compared with grade II tumors, grade IV tumors demonstrated higher blood volumes, but lower relative oxygen extraction and utilization. Tumor blood flow was variable, but was lower in the higher grade tumors. Rates of glucose utilization in tumor, calculated by using individually determined rate constants, were variable, and did not correlate with tumor size or tumor grade. Parietal tumors (n = 6) tended to have higher relative glucose utilization and blood flow, and lower relative oxygen extraction, when compared with frontal tumors (n = 4). Tumor pH differed significantly from the pH in contralateral brain (p less than 0.005); alkalotic pH values were consistently seen. These findings in and around cerebral gliomas studied before intervention differ from the results found in gliomas after exposure to radiation or chemotherapy.

Pharmacokinetics of superselective intra-arterial and intravenous [11C]BCNU evaluated by PET.

The pharmacokinetics of i.v. and superselective intra-arterial carbon-11 1,3-bis-(2-chloroethyl)-1-nitrosourea ([11C]BCNU) were directly compared for the first time in ten patients with recurrent gliomas using positron emission tomography (PET). Intra-arterial administration of [11C]BCNU achieved concentrations of the drug in the tumor that averaged 50 times higher than with a comparable i.v. dose. These preliminary results suggest that the degree of early metabolic trapping of BCNU in tumor correlates with the clinical response to this chemotherapy.

Early metabolic changes following chemotherapy of human gliomas in vivo demonstrated by phosphorus magnetic resonance spectroscopy.

The authors have used phosphorus magnetic resonance spectroscopy to monitor pH changes in malignant gliomas following treatment with intravenous and intra-arterial 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). Image-guided, localized phosphorus spectra of human gliomas in situ were obtained using a 1.5-T whole body combined imaging and spectroscopy system. Initial intravenous BCNU treatment was followed by a transient decrease of tumor intracellular pH by 0.15 +/- 0.03 pH units (mean +/- SD). Superselective intra-arterial administration of the same drug was followed by an increase of tumor intracellular pH by 0.15 +/- 0.6 pH units (mean +/- SD). These changes occurred prior to any changes on x-ray, computed tomography (CT), or magnetic resonance imaging (MRI). In addition to enhancing our understanding of the metabolic effects of BCNU, such changes may correlate with drug efficacy or toxicity and may be useful in guiding therapy in the future.

The cytolytic effect of L-2,4 diaminobutyric acid with malignant glioma cells and fibroblasts.

L-2,4-Diaminobutyric acid (DABA), an amino acid analogue, produced a cytolytic effect with a human glioma cell line, SKMG-1, and normal human fibroblasts. The concentrations of DABA necessary to reduce the cell count to 50% of control (LD50) following a 24-h incubation at 37 degrees C were 12.5 mM for the human fibroblasts and 20 mM for the glioma cell line. The concentrations of DABA necessary to produce an LD50 after a 48-h incubation at 37 degrees C were 10 mM for the human fibroblasts and 14 mM for the human glioma cell line. The cytolytic effect of DABA was similar in the absence or the presence of serum with the human glioma cell line. The cytolytic effect of 20 mM DABA was partially prevented by the presence of 5 mM methyl-AIB. DABA was not preferentially toxic to this human glioma cell line compared with normal fibroblasts.

The cytotoxicity of sarcosinamide chloroethylnitrosourea (SarCNU) and BCNU in primary gliomas and glioma cell lines: analysis of data in reference to theoretical peak plasma concentrations in man.

The cytotoxicity of a new compound, sarcosinamide chloroethylnitrosourea (SarCNU), was compared with that of the clinically available bis-chloroethylnitrosourea (BCNU) in 13 primary human gliomas and in 3 human glioma cell lines using the Human Tumor Cloning Assay (HTCA). At concentrations less than or equal to 16 micrograms/ml, SarCNU reduced the growth to less than or equal to 30% of control in 11 of 13 primary gliomas. At similar concentrations, BCNU produced a comparable cytotoxic effect in 6 out of 13 specimens. At concentrations less than or equal to 16 micrograms/ml, BCNU reduced colony growth to less than or equal to 30% of control in all three glioma cell lines and SarCNU produced the same effect in only one glioma cell line. A recently described statistical model, which employs the LD50 dose of new agents in mice, was used to estimate the achievable peak plasma concentration (PPC) of SarCNU. The calculated PPC for SarCNU was found to be 14.8 micrograms/ml compared with 2 micrograms#ml for BCNU. A reevaluation of the cytotoxic activities of SarCNU and BCNU at concentrations approximating their respective PPCs revealed that SarCNU reduced the growth to less than or equal to 30% of control in one cell line at a concentration below its PPC. In contrast, BCNU exhibited similar toxicity in each cell line only at concentrations exceeding its PPC of 2 micrograms/ml. In the case of the primary gliomas, SarCNU was active (less than or equal to 30% of control) in ten tumors at concentrations less than or equal to 14.8 micrograms/ml, whereas BCNU was active in only one glioma at a concentration less than or equal to 2 micrograms/ml. The results suggest that SarCNU should be more active than BCNU against human gliomas, provided that the statistical model used has correctly estimated the PPC of SarCNU.

Utilization of the HTSCA and CFU-C assay to identify two new 2-chloroethylnitrosourea congeners of amino acid amides with increased in vitro activity against human glioma compared with BCNU.

AspCNU and SarCNU are two amino acid amide congeners (L-asparaginamide and sarcosinamide congeners) of chloroethylnitrosoureas. The in vitro myelotoxicity of these agents compared with BCNU at 1-8 micrograms/ml was determined in bone marrow cells from normal volunteers in the CFU-C assay. AspCNU and SarCNU were significantly (P less than 0.05) less myelotoxic than BCNU at equivalent microgram concentrations. SarCNU or AspCNU at 3 micrograms/ml demonstrate equivalent in vitro myelotoxicity to BCNU 1 microgram/ml. We used the human tumor stem cell assay (HTSCA) to investigate in vitro antitumor activity. We obtained four specimens of malignant glioma and one specimen of meningioma from patients not previously treated with chemotherapy. AspCNU and SarCNU were significantly (P less than 0.05) more active than BCNU at 1-3 micrograms/ml concentrations in the HTSCA in all four malignant glioma specimens. In the one meningioma specimen, BCNU was significantly (P less than 0.05) more active than either AspCNU or SarCNU at all concentrations studied. These results suggest that AspCNU or SarCNU at doses that should produce less myelotoxicity than BCNU may be more active than BCNU against gliomas.

Neurosurgery at the Montreal Neurological Institute and McGill University Hospitals.

For the past 60 years, the Montreal Neurological Institute and Hospital and three associated McGill University teaching hospitals have provided a broad course of instruction in neurosurgery and the related neurosciences. This integrated program offers a wealth of experience in adult and pediatric neurosurgery, based on a total of 140 beds, covering a full range of general and subspecialty neurosurgery. The institute, recognized for many years as a world center for epilepsy surgery, has traditional strengths in the treatment of brain tumors and cerebrovascular and spinal disorders; it has been at the cutting edge of brain imaging in all modalities applied to neurosurgical diagnosis and cerebral localization, including three-dimensional monitoring in the operating room. New approaches to stereotactic procedures have been developed in conjunction with imaging technology, including functional neurosurgery and the versatile McGill double rotation method for radiosurgery with a linear accelerator. Experience in managing trauma, pediatric cases, and general neurosurgical problems is gained at the Montreal General Hospital, the Montreal Children's Hospital, and the Jewish General Hospital. Well-established research units, including burgeoning groups in neurogenetics, molecular neurobiology, and neural regeneration, provide a wide variety of academic opportunities to provide trainees with a sound basis for coping with the rapidly advancing field of neurosurgery.

Surgical treatment of intractable epilepsy associated with schizencephaly.

With the advent of magnetic resonance imaging, there has been an increased recognition of schizencephaly during life, especially in epileptic patients. We report our experience with the assessment and treatment of three patients with medically intractable seizures associated with this condition. The three men were aged 24 to 37 years. Two had delayed developmental milestones and hemiparesis or hemiplegia. One had normal development and a normal neurological examination. Seizures began between the ages of 15 and 19 years and lasted for 5 to 22 years before surgery. All had partial simple or generalized seizures with predominant electroencephalographic and electrocorticographic epileptic activity localized to temporal and frontal lobes on the side of the lesion. Neuropsychological assessment indicated widespread dysfunction maximal at the areas of predominant electroencephalographic abnormality. Magnetic resonance imaging demonstrated anterior parasagittal, parietal, and Rolandic cerebral clefts, with ventricular diverticuli, gray matter heterotopia, polymicrogyria, and a true agenesis of the corpus callosum in individual patients. The patients underwent temporal (one patient) and frontotemporal (two patients) lobectomies without additional neurological deficits or neuropsychological deterioration. Postoperative follow-up showed reduction in seizure frequency. We conclude that the surgical treatment of epilepsy is well tolerated in such patients, and their seizures can be alleviated by resection of epileptogenic areas.

Diagnostic potential of magnetic resonance in cases of foramen magnum meningiomas.

Meningioma is a histologically benign tumor that is second in frequency only to gliomas among primary intracranial tumors. Its extracerebral development and generally clear demarcation from the brain does not make it easier to detect on magnetic resonance (MR) scanning. Only 2 to 3% of meningiomas occur in the foramen magnum, and these tumors are often clinically misdiagnosed. We discuss four cases of foramen magnum meningioma, with emphasis on MR scanning.