Analysis of femtomole concentrations of alpha-ketoisocaproic acid in brain tissue by precolumn fluorescence derivatization with 4,5-dimethoxy-1,2-diaminobenzene.

An extremely sensitive fluorimetric HPLC method used to determine in vivo cerebral alpha-ketoisocaproic acid (KIC) concentration with less than 2 g of tissue (e.g., a single rat brain, approximately 1.8 g) is reported. Removal of unwanted lipids and amino acids and isolation of alpha-ketoacids in an optimal derivatization buffer are achieved by C18 solid-phase extraction of the acid-soluble fraction of brain tissue. Quantitation of KIC to the femtomole level is accomplished by reversed-phase HPLC using 4,5-dimethoxy-1,2-diaminobenzene precolumn fluorescence derivatization and on-line fluorescence detection. These techniques are applicable to femtomole quantitation of other alpha-ketoacids in various tissue and blood matrices. Combination of this fluorimetric method with simple nonchromatographic procedures to measure in vivo cerebral [1-14C]alpha-ketoisocaproic acid radioactivities in tissue provides estimates of specific activities.

Comparative in vivo metabolism of 6-[18F]fluoro-L-dopa and [3H]L-dopa in rats.

In vivo double-label experiments in rats were designed to correlate the peripheral and cerebral metabolism of 6-[18F]fluoro-L-DOPA [( 18F]FDOPA) with that of [3H]L-DOPA. Authentic samples of the major [18F]FDOPA metabolites were synthesized to identify the 18F-labeled metabolites. After carbidopa pretreatment and intravenous administration of the compound, the products of peripheral metabolism in plasma were analyzed at times from 3 to 60 min. In the periphery, amine conjugates were detected but they accounted for less than 15% of the total radioactivity; the major metabolites were 3-O-methyl-6-[18F]fluoro-L-DOPA and 3-O-methyl-[3H]L-DOPA. The rate and extent of 3-O-methylation of [18F]FDOPA exceeded that of [3H]L-DOPA. Both 3-O-methylated products entered the striatum and cerebellum where they contributed significant but uniform activity. Analysis of cerebral metabolism in these structures indicated a linear accumulation of total radioactivity: a striatum/cerebellum ratio of 2 was observed by 60 min. 6-[18F]Fluorodopamine (35%) and [3H]dopamine (55%) were the major metabolites formed in the striatum: however, the methylated [18F]FDOPA and [3H]DOPA products of predominantly peripheral origin represented 55% (18F) and 35% (3H) of the total radioactivity respectively. Other [3H]dopamine metabolites and their 18F-labeled analogs represented less than 10-15% at all times analyzed. The cerebellum radioactivity was composed only of [18F]FDOPA, [3H]DOPA and their 3-O-methylated products. These data will serve as the basis for the development of kinetic models of [18F]FDOPA metabolism that can be applied to the evaluation of central dopamine biochemistry with positron emission tomography in humans.

The effects of carbidopa on the metabolism of 6-[18F]fluoro-L-dopa in rats, monkeys and humans.

The effects of carbidopa on the peripheral metabolism of 6-[18F]fluoro-L-DOPA (FDOPA) were characterized in the rat, monkey and human along with its effects on cerebral FDOPA metabolism in the rat. After carbidopa pretreatment, FDOPA plasma metabolite profiles in all three species revealed extensive metabolism of FDOPA to 3-0-methyl-6-[18F]-fluoro-L-DOPA (3-OMFD). In humans, there were significant increases in FDOPA plasma levels for 30 min and in 3-OMFD levels for 120 min after FDOPA administration. 6-[18F]Fluorodopamine sulfate (FDA-sulfate) and [18F]fluoro-homovanillic acid (FHVA) levels were decreased, while at all times, free 6-[18F]-fluorodopamine (FDA) and 6-[18F]-3-4 dihydroxy-phenylacetic acid (FDOPAC) were not detected. In rat brain, the FDOPA metabolite profile at 30 min was significantly altered by carbidopa pretreatment; increases were noted for striatum FDA (700%) and 3-OMFD (230%), and for cerebellum FDOPA (370%) and 3-OMFD (300%). Thus, carbidopa pretreatment increased FDOPA plasma levels for a given FDOPA dose and essentially restricted peripheral FDOPA metabolism to 3-OMFD formation. The increase in FDOPA bioavailability to the brain resulted in greater selective FDA accumulation in striatum. As such, carbidopa pretreatment for FDOPA-positron emission tomography studies will significantly increase the amount of radioactivity that can be attributable to FDA in cerebral regions of interest.

3-(2'-[18F]fluoroethyl)spiperone: in vivo biochemical and kinetic characterization in rodents, nonhuman primates, and humans.

3-(2'-[18F]fluoroethyl)spiperone (FESP), a recently developed dopamine D2-receptor binding radiopharmaceutical, was used for dynamic characterization of dopamine-receptor binding in Macaca nemestrina monkeys and humans with positron emission tomography (PET). FESP in vitro binding properties to the dopamine receptor (IC50 = 1.5 nM) are similar to those of spiperone. Serial PET scans in monkeys after intravenous bolus injection of FESP revealed specific radioactivity accumulation in striatum (rich in dopamine D2-receptors), whereas radioactivity concentration declined after 20 min in frontal cortex (serotonin receptors) and more rapidly in cerebellum (nonspecific binding). Specific dopamine D2-receptor binding was saturated with increasing concentrations of radioligand (specific activity range: 1-10,000 Ci/mmol), was stereospecifically blocked with (+)butaclamol (0.5 mg/kg), and showed only partial displacement with spiperone (200 micrograms/kg, i.v. administration 90 min after FESP injection). From PET experiments with FESP in humans, it is possible to visualize accumulation of radioactivity in striatum in a manner similar to that observed in monkeys and, ex vivo, in rodents (adult male Sprague-Dawley rats). Biochemical analyses in rat brain revealed that the activity (approximately 90%) in striatum was unmodified FESP up to 4 h after injection. On the other hand, FESP was metabolized peripherally (rat greater than monkey greater than human), with only 11% of plasma radioactivity remaining as intact FESP in rodents and 54% in humans after 2 h. Based on these interspecies scaling pharmacokinetic data, it is unequivocal that FESP peripheral metabolites do not significantly contribute to the accumulated radioactivity in striatal tissue. Therefore, it is concluded that FESP is suitable for the quantitative estimation of dopamine D2-receptor sites using PET.

4-[18F]fluoro-L-m-tyrosine: an L-3,4-dihydroxyphenylalanine analog for probing presynaptic dopaminergic function with positron emission tomography.

4-[18F]Fluoro-L-m-tyrosine (FMT), a biochemical probe of striatal dopaminergic function, has been synthesized as an L-3,4-dihydroxyphenylalanine analog for positron emission tomography. Biochemical characterization of this compound in the rat 30 min after intrajugular administration indicated that in the brain, selective decarboxylation occurred in the striatum, with the formation of 4-fluoro-3-hydroxyphenylethylamine and its metabolites. Positron emission tomography analysis of brain tissue in monkeys (Macaca nemestrina) after intravenous injection of FMT revealed a true time-dependent, specific accumulation of radioactivity in striatum, with a striatum/cerebellum (nonspecific) ratio of 4 at 180 min. Peripheral metabolism accounted for less than 40% of the total radioactivity in arterial blood samples after 120 min. The amino acid remained as the major component throughout the period of investigation (n = 3; 5 min, 95%; 10 min, 85%; 30 min, 67%; 60 min, 62%; 120 min, 60%), with a plasma clearance t 1/2 of 112 min. 3-O-Methylated metabolites were not observed. The substrate specificity of FMT, coupled with its limited in vivo peripheral metabolism, makes it a useful, new biochemical probe for in vivo, noninvasive evaluation of central dopaminergic mechanisms.