Determination of extracellular and intracellular enrichments of [1-(13)C]-alpha-ketoisovalerate using enzymatically converted [1-(13)C]-valine standardization curves and gas chromatography-mass spectrometry.

We report a validated method for the determination of extra- and intracellular [1-(13)C]-alpha-ketoisovalerate ([1-(13)C]-KIV) enrichments by gas chromatography-mass spectrometry. Standardization curves were prepared by enzymatic oxidation of [1-(13)C]-valine enriched standards of known composition. Slopes of [1-(13)C]-valine standardization curves (mean+/-SD: 0.99+/-0.02, n=5) and [1-(13)C]-KIV standardization curves (mean+/-SD: 0.98+/-0.01, n=7) were not significantly different. The method was applied for the determination of [1-(13)C]-KIV enrichments in plasma and tissues during [1-(13)C]-valine infusion in a piglet. [1-(13)C]-KIV enrichment could be determined+/-0.1 MPE (C.V. 1%), and extracellular [1-(13)C]-KIV enrichment was a reliable estimate of intracellular (skeletal muscle, bone growth plate) [1-(13)C]-KIV enrichment.     

Estimating gluconeogenesis by NMR isotopomer distribution analysis of [13C]bicarbonate and [1-13C]lactate

The gluconeogenic contribution to glucose production in livers isolated from rats fasted for 24 h was determined by 13C-NMR isotopomer distribution analysis of secreted glucose enriched from 99% [13C]bicarbonate (n = 4) and 99% [1-13C]lactate (n = 4). Experiments with 3% 2H2O were also performed, allowing the gluconeogenic contribution to be measured by the relative 2H enrichments at positions 5 and 2 of glucose. From 13C-NMR analyses, the contribution of gluconeogenesis to glucose output was estimated to be 93 +/- 3% for [13C]bicarbonate perfusion and 91 +/- 3% for [1-13C]lactate perfusion, in good agreement with the 2H-NMR analysis of the gluconeogenic contribution to glucose production (100 +/- 1% and 99 +/- 1%, respectively) and consistent with the expected negligible contribution from glycogenolysis. These results indicate that 13C-NMR analysis of glucose 13C-isotopomer distribution from either [13C]bicarbonate or [1-13C]lactate precursor provides realistic estimates of the gluconeogenic contribution to hepatic glucose output.     

Measurement of leucine metabolism in man from a primed, continuous infusion of L-[1-3C]leucine

Leucine metabolism in vivo can be determined from a primed, continuous infusion of L-[1-13C]leucine by measuring, at isotopic steady state, plasm [-13C]leucine enrichment, expired 13CO2 enrichment, and CO2 production rate. With an appropriate priming dose of L-[1-13C]leucine and NaH13CO3, isotopic steady state is reached in less than 2 h, and the infusion is completed in 4 h. The method can determine rates of leucine turnover, oxidation, and incorporation into protein with typical relative uncertainties of 2, 10, and 4%, respectively. The method requires no more than 1 ml of blood and uses stable isotope rather than radioisotope techniques. Thus, the method is applicable to studies of human beings of all ages. L-[1-13C]leucine may be infused with a second amino acid labeled with 15N for simultaneous determination of the kinetics of two amino acids.     

Simultaneous determination of methionine and total homocysteine in human plasma by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method for the simultaneous determination of methionine and total homocysteine in human plasma is described. DL-[2H4]Methionine and DL-[2H8]homocystine were used as internal standards. The method involved reduction of the disulfide bond with dithiothreitol, purification by cation-exchange chromatography using a BondElut SCX cartridge and derivatization with isobutyl chlorocarbonate in water-ethanol-pyridine. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of N(O,S)-isobutyloxycarbonyl ethyl ester (IBC-OEt) derivatives for methionine and [2H4]methionine, respectively, and the fragment ions ([M+H-COOisoBu-COOEt]+) for TBC-OEt derivatives for homocysteine and [2H4]homocysteine, respectively. The sensitivity, specificity, accuracy and precision of the method were demonstrated to be satisfactory for measuring concentrations of methionine and total homocysteine in human plasma.     

Integration of [U-13C]glucose and 2H2O for quantification of hepatic glucose production and gluconeogenesis

Glucose metabolism in five healthy subjects fasted for 16 h was measured with a combination of [U-13C]glucose and 2H2O tracers. Phenylbutyric acid was also provided to sample hepatic glutamine for the presence of 13C-isotopomers derived from the incorporation of [U-13C]glucose products into the hepatic Krebs cycle. Glucose production (GP) was quantified by 13C NMR analysis of the monoacetone derivative of plasma glucose following a primed infusion of [U-13C]glucose and provided reasonable estimates (1.90 +/- 0.19 mg/kg/min with a range of 1.60-2.15 mg/kg/min). The same derivative yielded measurements of plasma glucose 2H-enrichment from 2H2O by 2H NMR from which the contribution of glycogenolytic and gluconeogenic fluxes to GP was obtained (0.87 +/- 0.14 and 1.03 +/- 0.10 mg/kg/min, respectively). Hepatic glutamine 13C-isotopomers representing multiply-enriched oxaloacetate and [U-13C]acetyl-CoA were identified as multiplets in the 13C NMR signals of the glutamine moiety of urinary phenylacetylglutamine, demonstrating entry of the [U-13C]glucose tracer into both oxidative and anaplerotic pathways of the hepatic Krebs cycle. These isotopomers contributed 0.1-0.2% excess enrichment to carbons 2 and 3 and approximately 0.05% to carbon 4 of glutamine.     

Metabolism of D-[1,2-13C]glucose and alpha-D-[1,2-13C]glucose pentaacetate in tumoral pancreatic islet cells

Tumoral pancreatic islet cells of the RINm5F line were incubated, in groups of 25x106 cells each, for 120 min at 37 degrees C in media (5. 0 ml) containing either alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM) or both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM). In both cases, the amounts of 13C-enriched metabolites (D-glucose, L-lactate and acetate) and non-enriched metabolites (acetate) recovered in the incubation medium after incubation were close to the initial amount of esterified or non-esterified D-[1, 2-13C]glucose and acetate, respectively. The 13C-enriched metabolites corresponded mainly to double-labelled D-[1, 2-13C]glucose, L-[2,3-13C]lactate and [1,2-13C]acetate. The output of L-[2,3-13C]lactate and [1,2-13C]acetate was about 3-4 times lower in the cells exposed to alpha-D-[1,2-13C]glucose pentaacetate than in those incubated with unesterified D-[1,2-13C]glucose. These findings indicate that, despite extensive hydrolysis of alpha-D-[1, 2-13C]glucose pentaacetate in the RINm5F cells, the hexose moiety of the ester is less efficiently metabolized than unesterified D-[1, 2-13C]glucose tested at the same molar concentration (1.7 mM) in the presence of 8.5 mM acetate. Thus, a higher utilization of the hexose moiety of alpha-D-glucose pentaacetate than that of unesterified D-glucose, as previously documented in isolated pancreatic islets, represents a far-from-universal situation.     

Apparent Galactose Appearance Rate in Human Galactosemia Based on Plasma [C]Galactose Isotopic Enrichment

Determination of endogenous galactose formation in galactosemic subjects provides important information in understanding the etiology of the long-term complications. To accomplish this task a sensitive method for measurement of isotopic enrichment of plasma galactose was developed. The aldononitrile pentaacetate derivative of galactose was utilized for gas chromatography/mass spectrometry analysis. Using a phenyl-methylsilicone capillary column, adequate separation of galactose from glucose was obtained by temperature programming of the chromatography. The specific fragmentation pattern of m/z 212, 225, 314 from -[¹²C]galactose and m/z 213, 226, 315 from -[¹³C]galactose was used for the galactose enrichment measurement by atom percent excess (APE). There was good correlation between expected enrichment and determined APEs at galactose concentrations of 1, 2, and 5 μmol/L with a coefficient of variation ranging from 0.22 to 7.17%. The method provides an accurate estimation of plasma [¹³C]galactose enrichment from which the galactose production rate can be calculated. The steady-state plasma -[¹³C]galactose isotopic enrichment of three individuals with galactosemia, two males ages 33 and 13, and one female age 9, during constant infusion of -[¹³C]galactose was 55, 41, and 55%, allowing the estimation of the apparent galactose appearance rate of 0.62, 1.09, and 0.82 mg/kg/h, respectively. The reanalysis of three previous studies by the present method found that APE values determined by the method then employed, butylboronate acetate derivatization, were systemically lower than those determined with aldononitrile pentaacetate derivatization, making for an overestimation of the apparent galactose appearance rate. The small plasma sample volumes needed make it feasible to perform these studies in infants and young children with galactosemia.     

Metabolism of D-[1-(13)C]fructose, D-[2-(13)C]fructose, and D-[6-(13)C]fructose in rat hepatocytes incubated in the presence of H(2)O or D(2)O

Isolated hepatocytes from fed rats were exposed for 120 min to D-[1-(13)C]fructose, D-[2-(13)C]fructose, or D-[6-(13)C]fructose in the presence of H(2)O or D(2)O. The identification and quantification of (13)C-enriched metabolites (D-glucose, L-lactate) in the incubation medium and the measurement of their deuterated isotopomers indicated that the ketohexose was phosphorylated predominantly at the intervention of fructokinase and that the majority of the D-glyceraldehyde molecules generated from d-fructose 1-phosphate were further metabolized, e.g., after phosphorylation to D-glyceraldehyde 3-phosphate. It is proposed that the present procedure may help to further characterize the regulation of D-fructose metabolism in both hepatocytes and other cell types.     

NMR study on [1-13C]glucose metabolism in the rat brain during hypoxia

The metabolism of [1-13C]glucose in the rat brain during hypoxia was investigated by 13C NMR spectroscopy. Male Wistar rats, weighing 100-120g, were anesthetized with ketamine (50 approximately 75 mg/kg i.p.) and ventilated mechanically with a mixture of 30% oxygen, 69.5% nitrogen and 0.5% halothane. [1-13C]glucose (250 mg/kg) was infused twice, at 10 minute intervals, through the femoral vein. For the control group (n = 4), the oxygen concentration of the inspiratory gas was maintained at 30% by vol throughout the experiments. For the hypoxia group (n = 6), the oxygen concentration in the inspiratory gas was reduced to 6-7% (93-94% nitrogen) and maintained for 30 min following [1-13C]glucose infusion. 13C NMR spectra were measured by a gated proton-decoupling method without a nuclear Overhauser effect. The [1-13C]glucose infusion gave apparent signals of the C1 carbon in the alpha- and beta-anomers of [1-13C]glucose at 92.7 and 96.7 ppm, respectively. Signals of the C2, C3 and C4 carbon atoms in glutamate and/or glutamine (glx) also appeared at 55, 27 and 34 ppm, respectively. The intensity of glx-C2 and glx-C3 signals increased later than that of glx-C4. The time lag between the different glx signals may reflect the turnover rate of the TCA cycle. Under the hypoxic condition, the signal of C3 carbon in lactate appeared at 21 ppm and increased. The alpha-glucose signal diminished during hypoxia, whereas the beta-glucose signal kept its intensity. The difference in changes of the signal intensity between alpha- and beta-glucose suggests that alpha-glucose is consumed more than beta-glucose in the hypoxic brain.     

Rapid and highly sensitive method for the determination of verapamil, [2H7]verapamil and metabolites in biological fluids by liquid chromatography-mass spectrometry

A rapid and highly sensitive method for the determination of verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile+ ++] and [2H7]verapamil and their primary metabolites D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile], D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxy-phenyl)-6-methyl-2-iso-p ropyl-6-azaoctanitrile], D-702 [2-(3,4-dimethoxy-phenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopr opyl-6-azaoctanitrile], norverapamil [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile] and secondary metabolites D-620 [2-(3,4-dimethoxyphenyl)-5-amino-2-isopropylvaleronitrile], D-717 [2-(4-hydroxy-3-methoxyphenyl)-5-amino-2-isopropylvaleronitrile], and D-715 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxy-phenyl)-2-isopropyl-6-++ +azaoctanitrile] has been developed using high-performance liquid chromatography-electrospray mass spectrometry. D-832, the gallopamil analogue of D-617 and [2H3]norverapamil were used as internal standards. The analytes were extracted automatically from plasma and intestinal perfusate using end-capped CN- and C2 solid-phase extraction cartridges. Separation of the eight analytes was achieved on a LUNA C8 analytical column (150x2 mm I.D., 5 microm particle size) with 5 mM ammonium acetate-acetonitrile as the mobile phase run with a gradient from 70:30 to 40:60 and run times of 15 min. With the mass spectrometer operated in the selected-ion monitoring mode, the limits of quantification in plasma and intestinal perfusate were 1 pmol/ml for D-620, D-617, D-702, D-703, norverapamil, verapamil, and [2H7]verapamil and 2.5 pmol/ml for D-717 and D-715 using a sample size of 1 ml plasma and intestinal perfusate. The method described was successfully applied to the determination of verapamil, [2H7]verapamil and their metabolites in human plasma and intestinal fluid in pharmacokinetic studies.     

The effect of 13C enrichment in the glassing matrix on dynamic nuclear polarization of [1-13C]pyruvate

Dimethyl sulfoxide (DMSO) can effectively form a glassy matrix necessary for dynamic nuclear polarization (DNP) experiments. We tested the effects of (13)C enrichment in DMSO on DNP of [1-(13)C]pyruvate doped with trityl radical OX063Me. We found that the polarization build-up time τ of pyruvate in (13)C-labeled DMSO glassing solution is twice as fast as the unenriched DMSO while the nuclear magnetic resonance enhancement was unchanged. This indicates that (13)C-(13)C spin diffusion is a limiting factor in the kinetics of DNP in this system, but it has a minimal effect on the absolute value of polarization achievable for the target.     

Apparent galactose appearance rate in human galactosemia based on plasma [(13)C]galactose isotopic enrichment

Determination of endogenous galactose formation in galactosemic subjects provides important information in understanding the etiology of the long-term complications. To accomplish this task a sensitive method for measurement of isotopic enrichment of plasma galactose was developed. The aldononitrile pentaacetate derivative of galactose was utilized for gas chromatography/mass spectrometry analysis. Using a phenyl-methylsilicone capillary column, adequate separation of galactose from glucose was obtained by temperature programming of the chromatography. The specific fragmentation pattern of m/z 212, 225, 314 from d-[(12)C]galactose and m/z 213, 226, 315 from l-[(13)C]galactose was used for the galactose enrichment measurement by atom percent excess (APE). There was good correlation between expected enrichment and determined APEs at galactose concentrations of 1, 2, and 5 micromol/L with a coefficient of variation ranging from 0.22 to 7.17%. The method provides an accurate estimation of plasma [(13)C]galactose enrichment from which the galactose production rate can be calculated. The steady-state plasma l-[(13)C]galactose isotopic enrichment of three individuals with galactosemia, two males ages 33 and 13, and one female age 9, during constant infusion of l-[(13)C]galactose was 55, 41, and 55%, allowing the estimation of the apparent galactose appearance rate of 0.62, 1.09, and 0.82 mg/kg/h, respectively. The reanalysis of three previous studies by the present method found that APE values determined by the method then employed, butylboronate acetate derivatization, were systemically lower than those determined with aldononitrile pentaacetate derivatization, making for an overestimation of the apparent galactose appearance rate. The small plasma sample volumes needed make it feasible to perform these studies in infants and young children with galactosemia.     

Metabolism of D-[3-3H]glucose, D-[5-3H]glucose, D-[U-14C]glucose, D-[1-14C]glucose and D-[6-14C]glucose in pancreatic islets in an animal model of type-2 diabetes

This study aims at exploring specific aspects of D-glucose metabolism, so far not yet investigated, in pancreatic islets from adult control rats and animals (STZ rats) injected with streptozotocin during the neonatal period. The latter animals, which represent a current model of type-2 diabetes, displayed a lower body weight, higher plasma D-glucose concentration and lower insulinogenic index than control rats. The protein, DNA and insulin content were all also lower in islets prepared from STZ, rather than control rats. In the presence of 10.0 mM D-glucose, the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was also decreased in the islets from STZ rats. No significant difference between control and STZ rats was observed, however, in terms of the ratios between D-[3-3H]glucose and D-[5-3H]glucose utilization, between the generation of radioactive lactate from 14C-labelled D-glucose and tritiated D-glucose utilization and between D-[1-14C]glucose and D-[6-14C]glucose oxidation. These findings reinforce the view that the previously documented preferential impairment of the oxidative modality of glycolysis in islets from STZ rats contrasts with the absence of any major anomaly in other variables of D-glucose catabolism.     

Metabolism of [3-13C]-pyruvate by cysticerci of Taenia crassiceps

Carbon-13 decoupled ¹H spin-echo nuclear magnetic resonance (NMR) spectra, with and without ¹³C population inversion, of extracts of Taenia crassiceps cysticerci incubated in media containing [3-¹³C]-pyruvate showed ¹³C enrichment in alanine, lactate, acetate, succinate, and citrate. Labeling in the latter three metabolites provides substantial evidence that the malic enzyme reaction in this cestode also functions in the direction opposite to that in which it is normally portrayed. The direct passage of pyruvate from the cytosol to the mitochondrion is suggested by the greater percentage of ¹³C detected in acetate relative to succinate. Received: 8 July 1997 / Accepted: 19 December 1997     

Metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C]succinate) in hepatocytes from Goto-Kakizaki rats

The metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate) was examined in hepatocytes prepared from hereditarily diabetic Goto-Kakizaki rats. Over 120 min incubation in the presence of one of the two (13)C-labelled esters (2.5 mM), the output of (13)C-enriched glucose averaged 57.1 +/- 18.5 and 54.1 +/- 22.7 nmol per 10(6) cells, when expressed as [1,3-(13)C]glycerol and [2,3-(13)C] succinate equivalent, respectively. In the case of [1,3-(13)C]glycerol-1,2,3-tris(methyl-succinate), the molecules of glucose were symmetrically labelled. In the case of glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate), however, both the single-labelled and double-labelled isotopomers of glucose contained more (13)C atoms in their C(6)-C(5)-C(4) than C(1)-C(2)-C(3) moiety. These findings indicate that glycerol-1,2,3-tris(methylsuccinate), recently proposed as a novel insulinotropic tool for the treatment of non-insulin-dependent diabetes mellitus, is efficiently metabolized in hepatocytes from diabetic rats, the high rate of gluconeogenesis coinciding with channelling of D-glyceraldehyde-3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.     

Carbohydrate metabolism of the rat C6 glioma. An in vivo 13C and in vitro 1H magnetic resonance spectroscopy study

Surface coil 13C nuclear magnetic resonance (NMR) spectroscopy was used to investigate the in vivo carbohydrate metabolism of rat C6 gliomas during and after infusion with [1-13C] glucose. In vivo 1H-decoupled 13C NMR spectra of the glioma following infusion with [1-13C]glucose revealed the direct production of [3-13C]lactic acid, [1-13C]glycogen, and [4-13C], [3-13C], and [2-13C]glutamate/glutamine. Lactate levels of in vivo gliomas increased and reached steady state levels during [1-13C]glucose infusion, and decreased following termination of infusion. Complementary in vitro studies using supernatant media collected from C6 glioma cells incubated with media containing [1-13C] or [6-13C]glucose and glutamine were examined by 1H NMR spectroscopy. The [3-(13C/12C)]lactate ratios obtained from 1H spectra of supernatant media containing [1-13C]glucose revealed the percentage of glucose metabolized through the hexose monophosphate shunt to be 10.01 +/- 0.85% (n = 3), while similar measurements of media containing [6-13C]glucose and glutamine showed that glutaminolysis contributed 9.0 +/- 1.0% of total lactate production under these conditions. Enzymatic analysis of media determined lactate production to be 139 +/- 9 nmol per 10(6) cells per h (n = 4). These measurements demonstrate the ability of NMR to monitor brain tumor carbohydrate metabolism both in vitro and in vivo.     

A 13C NMR study on fluxes into the Krebs cycle of rabbit renal proximal tubular cells

Suspensions of rabbit renal proximal tubular (PCT) cells were incubated with [2-13C] and [3-13C]pyruvate. The perchloric acid extracts of the cell pellets were examined by 13C NMR. All experiments showed that enriched lactate, alanine, glutamate, and glutamine were the main metabolic intermediates, and that enrichment to a minor extent was found in the glutamate residue of glutathione (GSH). From these experiments, it could be deduced that PCT cells show a highly glycolytic activity, whereas enrichment of glucose exhibits gluconeogenesis. The estimation by 13C NMR of the ratio of the flux into the Krebs cycle via pyruvate carboxylase to the flux via pyruvate dehydrogenase is discussed. From incubations with 10 mM 13C-labelled pyruvate, we calculated from the relative enrichments of the glutamate carbon atoms that the ratio of pyruvate carboxylase to pyruvate dehydrogenase is 1.44 +/- 0.04 in rabbit renal proximal tubules.     

Autoradiography of [14C]paraquat or [14C]diquat in frogs and mice: accumulation in neuromelanin

The herbicide paraquat has been suggested as a causative agent for Parkinson's disease because of its structural similarity to a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which may induce a parkinsonism-like condition. MPTP as well as its metabolite 1-methyl-4-phenylpyridine have melanin affinity, and the parkinsonism-inducing potency of MPTP is much stronger in species with melanin in the nerve cells. Autoradiography of [3H]MPTP in experimental animals has shown accumulation in melanin-containing tissues, including pigmented neurons. In the present whole body autoradiographic study accumulation and retention was seen in neuromelanin in frogs after i.p. injection of [14C]paraquat or [14C]diquat. By means of whole body autoradiography of [14C]diquat in mice (a species with no or very limited amounts of neuromelanin) a low, relatively uniformly distributed level of radioactivity was observed in brain tissue. Accumulation of toxic chemical compounds, such as paraquat, in neuromelanin may ultimately cause lesions in the pigmented nerve cells, leading to Parkinson's disease.     

Simultaneous determination of endogenous and 13C-labelled cortisols and cortisones in human plasma by stable isotope dilution mass spectrometry

This study describes a capillary GC-MS method for the simultaneous determination of endogenous cortisol and cortisone and their 13C-labelled analogues, [1,2,4,19-13C4]cortisol (cortisol-13C4) and [1,2,4,19-13C4]cortisone (cortisone-13C4), in human plasma. [1,2,4,19-13C4,1,1,19,19,19-2H5]Cortisol (cortisol-13C4,2H5) and [1,2,4,19-13C4,1,1,19,19,19-2H5]cortisone (cortisone-13C4,2H5) were used as analytical internal standards. A double derivatization (bismethylenedioxy-pentafluoropropionate, BMD-PFP) with good GC behavior was employed for the GC-MS analysis of cortisol and cortisone. Quantitation was carried out by selected-ion monitoring of the molecular ions ([M]+*) of the BMD-PFP derivatives of cortisol and cortisone. The sensitivity limit of the present GC-MS-SIM method was found to be 150 pg per injection for cortisol (s/n=5.0) and 50 pg for cortisone (s/n=8.1). The within-day reproducibility in which the amounts of unlabelled and labelled cortisols and cortisones determined were in good agreement with the actual amounts added, the relative errors being less than 3.07%. The inter-assay coefficients of variation (C.V.) were less than 1.80% for unlabelled and labelled cortisols and cortisones.     

Autoradiographic visualisation of [3H]DTG binding to sigma receptors, [3H]TCP binding sites, and L-[3H]glutamate binding to NMDA receptors in human cerebellum.

The autoradiographic distributions of [3H]1,3-di-ortho-tolyguanidine ([3H]DTG), [3H]1-[1-(2-thienyl) cyclohexyl] piperidine ([3H]TCP) and L-[3H]glutamate were studied in the human cerebellum. [3H]DTG is a selective label for the sigma receptor, while L-[3H]glutamate binding was carried out under conditions selective for the N-methyl-D-aspartate (NMDA) receptor. [3H]TCP binding sites and sigma receptors showed marked enrichment in the Purkinje cell layer, while L-[3H]glutamate-labelled NMDA receptors showed virtually no binding in the Purkinje cell layer. The results confirm the existence of [3H]TCP binding sites which are not linked to NMDA receptors in the human cerebellum, having a distribution which is more similar to that of the haloperidol-sensitive sigma receptor.