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Purpose: With the recent developments in novel molecular targeted therapy such as immune-checkpoint blockades, serine/threonine-protein kinase B-Raf, and mitogen-activated protein kinase kinase inhibitors, the prognosis of advanced malignant melanoma has been improving. 5-S-cysteinyl-dopa (5-S-CD), a precursor of pheomelanin, has been previously revealed to be a useful biomarker for advanced-stage malignant melanoma, especially in patients with distant metastases. Here, we aimed to assess and compare the utility of serum 5-S-CD and lactate dehydrogenase levels as markers for predicting the effects of nivolumab in advanced malignant melanoma. Methods: Baseline serum 5-S-CD and lactate dehydrogenase levels in patients with unresectable stage IIIC and IV malignant melanoma treated with nivolumab ( n = 21) were analyzed to determine their utility as predictive markers for survival. We also analyzed the prognostic value of these markers among patients with only stage IV malignant melanoma ( n = 17). Results: Our analysis showed that patients with baseline serum 5-S-CD levels >25.0 nmol/L had significantly poor prognosis. In contrast, serum lactate dehydrogenase levels at the upper limit of the normal range did not exhibit such changes. Conclusions: Serum 5-S-CD levels have the potential to be an excellent predictive marker for the efficacy of nivolumab therapy in patients with advanced malignant melanoma.

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.

The Patlak graphical analysis (PGAREF) for quantification of irreversible tracer binding with a reference tissue model was approximated by a dual time point imaging approach (DTPREF). The DTPREF was applied to 18 [18F]-FDOPA brain scans using the occipital cortex as reference region (DTPOCC) and compared to both PGAOCC and striatal-to-occipital ratios (SOR). Pearson correlation analysis and Bland–Altman plots showed an excellent correlation and good agreement between DTPOCC and PGAOCC, while correlations between SOR and PGAOCC were consistently lower. Linear discriminant analysis (LDA) demonstrated a similar performance for all methods in differentiating patients with Parkinson’s disease (PD) from healthy controls (HC). Specifically for [18F]-FDOPA brain imaging, these findings validate DTPOCC as an approximation for PGAOCC, providing the same quantitative information while reducing the acquisition time to two short static scans. For PD patients, this approach can greatly improve patient comfort while reducing motion artifacts and increasing image quality. In general, DTPREF can improve the clinical applicability of tracers with irreversible binding characteristics when a reference tissue is available.

O-methyl metabolite (L-[ β-11C]OMD) of 11C-labeled L-3,4-dihydroxyphenylalanine (L-[ β-11C]DOPA) can penetrate into brain tissue through the blood–brain barrier, and can complicate the estimation of dopamine synthesis capacity by positron emission tomography (PET) study with L-[ β-11C]DOPA. We evaluated the impact of L-[ β-11C]OMD on the estimation of the dopamine synthesis capacity in a human L-[ β-11C]DOPA PET study. The metabolite correction with mathematical modeling of L-[ β-11C]OMD kinetics in a reference region without decarboxylation and further metabolism, proposed by a previous [18F]FDOPA PET study, were implemented to estimate radioactivity of tissue L-[ β-11C]OMD in 10 normal volunteers. The component of L-[ β-11C]OMD in tissue time-activity curves (TACs) in 10 regions were subtracted by the estimated radioactivity of L-[ β-11C]OMD. To evaluate the influence of omitting blood sampling and metabolite correction, relative dopamine synthesis rate ( kref) was estimated by Gjedde–Patlak analysis with reference tissue input function, as well as the net dopamine synthesis rate ( Ki) by Gjedde–Patlak analysis with the arterial input function and TAC without and with metabolite correction. Overestimation of Ki was observed without metabolite correction. However, the kref and Ki with metabolite correction were significantly correlated. These data suggest that the influence of L-[ β-11C]OMD is minimal for the estimation of kref as dopamine synthesis capacity.

Longitudinal measurements of dopamine (DA) uptake and turnover in transgenic rodents may be critical when developing disease-modifying therapies for Parkinson's disease (PD). We demonstrate methodology for such measurements using [18F]fluoro-3,4-dihydroxyphenyl- L-alanine ([18F]FDOPA) positron emission tomography (PET). The method was applied to 6-hydroxydopamine lesioned rats, providing the first PET-derived estimates of DA turnover for this species. Control ( n = 4) and unilaterally lesioned ( n = 11) rats were imaged multiple times. Kinetic modeling was performed using extended Patlak, incorporating a kloss term for metabolite washout, and modified Logan methods. Dopaminergic terminal loss was measured via [11C]-(+)-dihydrotetrabenazine (DTBZ) PET. Clear striatal [18F]FDOPA uptake was observed. In the lesioned striatum the effective DA turnover increased, shown by a reduced effective distribution volume ratio ( EDVR) for [18F]FDOPA. Effective distribution volume ratio correlated ( r > 0.9) with the [11C]DTBZ binding potential ( BPND). The uptake and trapping rate ( kref) decreased after lesioning, but relatively less so than [11C]DTBZ BPND. For normal controls, striatal estimates were kref = 0.037 ± 0.005 per minute, EDVR = 1.07 ± 0.22 and kloss = 0.024 ± 0.003 per minute (30 minutes turnover half-time), with repeatability (coefficient of variation) ≤11%. [18F]fluoro-3,4-dihydroxyphenyl- L-alanine PET enables measurements of DA turnover in the rat, which is useful for developing novel therapies for PD.

A home-made nano-desorption electrospray ionization (nano-DESI) device and the kinetic method were tested in chiral analysis of model clinical samples containing enantiomers of one of three pharmaceutically important compounds: dihydroxyphenylalanine (DOPA), ephedrine and ibuprofen. The initial evaluation of chiral systems was carried out by direct infusion of solution mixtures (analyte/central metal/chiral reference ligand) to a standard electrospray ionization (ESI) source. Cu(II) was used as a central metal for all analytes, l-phenylalanine was applied as a chiral reference ligand for DOPA, whereas l-tryptophan was used for the other two analytes. Then, the ESI source was substituted by a nano-DESI source and dried spots of 1 μL samples of whole human blood spiked with individual drugs were successfully analyzed without any pre-treatment. Irrespective of a laborious initial nano-DESI set-up, the combination of the kinetic method with nano-desorption electrospray has, for the first time, been demonstrated as a promising tool for chiral analysis of drugs in blood samples.

Deficiency of phenylalanine hydroxylase activity in phenylketonuria (PKU) causes an excess of phenylalanine (Phe) throughout the body, predicting impaired synthesis of catecholamines in the brain. To test this hypothesis, we used positron emission tomography (PET) to measure the utilization of 6-[18F]fluoro-l-dopamine (FDOPA) in the brain of adult patients suffering from PKU and in healthy controls. Dynamic 2-h long FDOPA emission recordings were obtained in seven adult PKU patients (five females, two males; age: 21 to 27 years) with elevated serum Phe levels, but lacking neurologic deficits. Seven age-matched, healthy volunteers were imaged under identical conditions. The utilization of FDOPA in striatum was calculated by linear graphical analysis ( k3S, min−1), with cerebellum serving as a nonbinding reference region. The time to peak activity in all brain time—radioactivity curves was substantially delayed in the PKU patients relative to the control group. The mean magnitude of k3S in the striatum of the PKU patients (0.0052±0.0004 min−1) was significantly lower than in the control group (0.0088±0.0009 min−1) ( P3S. The unidirectional clearance of FDOPA to brain was impaired in adult patients suffering from PKU, presumably reflecting the competitive inhibition of the large neutral amino acid carrier by Phe. Assuming this competition to be spatially uniform, the relationship between striatum and cerebellum time—activity curves additionally suggests inhibition of DOPA efflux, possibly also due to competition from Phe. The linear graphical analysis shows reduced k3S in striatum, indicating reduced DOPA decarboxylase activity.

Background: Information on the significance of an elevated urinary dopamine is limited and can lead to misinterpretation of the cause of such a finding. This laboratory-based study examines the associations with elevated dopamine gathered from a significant number of patients. Methods: The urine catecholamine and metabolite results of specimens (analysed by HPLC-ECD) from 5933 adults and 467 children were examined retrospectively over a 57-month period. Those with elevated dopamine were identified and the explanation for this finding was sought. Results: In adults, the conditions associated with an elevated dopamine were: urine over-collection; drug effects (including those due to intravenous dopamine, L-dopa, methyldopa, clozapine, antidepressants and metoclopramide); clinical effects (including those due to phaeochromocytoma, carcinoid tumour and pregnancy). In children, high urine dopamine was found in cases of neuroblastoma, Costello syndrome, leukaemia, phaeochromocytoma, Menkes disease and rhabdomyosarcoma of the bladder. Conclusions: A high urine dopamine was found in

Conventional methods for the graphical analysis of 6-[18F]fluorodopa (FDOPA)/positron emission tomography (PET) recordings ( Kappin) may be prone to negative bias because of oversubtraction of the precursor pool in the region of interest, and because of diffusion of decarboxylated FDOPA metabolites from the brain. These effects may reduce the sensitivity of FDOPA/PET for the detection of age-related changes in dopamine innervations. To test for these biasing effects, we have used a constrained compartmental analysis to calculate the brain concentrations of the plasma metabolite 3- O-methyl-FDOPA (OMFD) during 120 mins of FDOPA circulation in healthy young, healthy elderly, and Parkinson's disease subjects. Calculated brain OMFD concentrations were subtracted frame-by-frame from the dynamic PET recordings, and maps of the FDOPA net influx to brain were calculated assuming irreversible trapping ( Kapp). Comparison of Kappin and Kapp maps revealed a global negative bias in the conventional estimates of FDOPA clearance. The present OMFD subtraction method revealed curvature in plots of Kapp at early times, making possible the calculation of the corrected net influx ( K) and also the rate constant for diffusion of decarboxylated metabolites from the brain ( kloss). The effective distribution volume (EDV2; K/ kloss) for FDOPA, an index of dopamine storage capacity in brain, was reduced by 85% in putamen of patients with Parkinson's disease, and by 58% in the healthy elderly relative to the healthy young control subjects. Results of the present study support claims that storage capacity for dopamine in both caudate and putamen is more profoundly impaired in patients with Parkinson's disease than is the capacity for DOPA utilization, calculated by conventional FDOPA net influx plots. The present results furthermore constitute the first demonstration of an abnormality in the cerebral utilization of FDOPA in caudate and putamen as a function of normal aging, which we attribute to loss of vesicular storage capacity.

The antipsychotic efficacy of aripiprazole is not generally associated with extrapyramidal symptoms, cardiovascular effects, sedation or elevations in serum prolactin that characterize typical or atypical antipsychotics. The aim of this study was to clarify the mechanism of action of aripiprazole that underlies its favourable clinical profiles. The preclinical efficacy and side-effect profiles of aripiprazole were evaluated usingseveral pharmaco-behavioural test systems in mice and rats, both in vivo and ex vivo, and compared with those of other conventional and atypical antipsychotics. Each of the antipsychotics induced catalepsy and inhibited apomorphine-induced stereotypy. The catalepsy liability ratios for these drugs were 6.5 for aripiprazole, 4.7 for both olanzapine and risperidone. The ptosis liability ratios for aripiprazole, olanzapine and risperidone were 14, 7.2 and 3.3, respectively. Aripiprazole slightly increased DOPA accumulation in the forebrain of reserpinised mice, reduced 5-HTP accumulation at the highest dose and exhibited a weaker inhibition of 5-methoxy-N,N-dimethyl-tryptamine-induced head twitches. Aripiprazole did not inhibit physostigmine- or norepinephrine-induced lethality in rats. In conclusion, aripiprazole shows a favourable preclinical efficacy and side-effect profile compared to a typical antipsychotics. This profile may result from its high affinity partialagonist activity at D2 and 5-HT1A receptors and its antagonism of 5-HT2A receptors.

Parkinson disease is characterized by the loss of dopaminergic neurons, thus decreasing the system's ability to produce and store dopamine (DA). Such ability is often investigated using 18F-fluorodopa (FD) positron emission tomography. A commonly used model to investigate the DA synthesis and storage rate is the modified Patlak graphical approach. This approach allows for both plasma and tissue input functions, yielding the respective uptake rate constants Ki and Kocc. This method requires the presence of an irreversible compartment and the absence of any nontrapped tracer metabolite. In the case of Kocc, this last assumption is violated by the presence of the FD metabolite 3- O-methyl-[18F]fluoro-dopa (3OMFD), which makes the Kocc evaluation susceptible to a downward bias. It was found that both Ki and Kocc are influenced by DA loss and thus are not pure measures of DA synthesis and storage. In the case of Kocc, the presence of 3OMFD exacerbates the effect of DA egress, thus introducing a disease-dependent bias in the Kocc determination. These findings imply that Ki and Kocc provide different assessments of disease severity and that, as disease progresses, Ki and especially Kocc become more related to DA storage capacity and less to the DA synthesis rate.

Occlusal disharmonies have classically been thought to be involved in the etiopathogenesis of bruxism, as have, more recently, alterations in central neurotransmission, particularly dopaminergic neurotransmission. However, the connection between these two factors has still not been established. In this study, we assessed the effects of diverse occlusal disharmonies, maintained for either 1 day or 14 days, on neurochemical indices of dopaminergic and noradrenergic activity in the striatum, frontal cortex, and hypothalamus of the rat. The in vivo activity of tyrosine hydroxylase, determined as the accumulation of 3,4-dihydroxyphenylalanine (DOPA), 30 min after the administration of 3-hydroxybenzylhydrazine, a DOPA decarboxylase inhibitor, and dopamine and noradrenaline contents were quantified by high-performance liquid chromatography with electrochemical detection. The wearing of an acrylic cap on both lower incisors for 1 day induced a significant increase in DOPA accumulation in the regions analyzed, with parallel increases in dopamine levels in the hypothalamus and dopamine and noradrenaline in the frontal cortex. After the cap was maintained for 14 days, DOPA accumulation tended to return to control values, except in the left striatum, thereby causing an imbalance between hemispheres. In contrast, 1 or 14 days after the lower left and the upper right incisors were cut, less pronounced changes in catecholaminergic neurotransmission were found in the brain areas studied. Moreover, the cutting of one lower incisor did not modify either DOPA accumulation or dopamine and noradrenaline contents in the striatum or hypothalamus. These results provide experimental evidence of a modulation of central catecholaminergic neurotransmission by occlusal disharmonies, being dependent on the nature of the incisal alteration and on the time during which it was maintained.

Variations in the cerebellum to plasma ratio at late times in 6-[18F]fluoro-l-DOPA studies are shown to be consistent with competitive binding of large neutral amino acids for a common transporter in the blood—brain barrier and the stability of brain tissue large neutral amino acid level in the presence of plasma level changes. The distribution volume of an inert large neutral amino acid can be estimated from plasma and tissue large neutral amino acid levels and apparent half-saturation concentrations ( Km) of the transporter in the blood—brain barrier. Stability of brain large neutral amino acid levels is supported by literature findings and can be explained by high saturation of the large neutral amino acid transporter at physiologic conditions.

Three-dimensional (3D) positron emission tomography (PET) is attractive for [18F]fluoro-DOPA studies, since the sensitivity improvement is maximal for radioactive sources located in central planes, which is usually the case for the human striata. However, the image quantitation in that mode must be assessed because of the nearly threefold increase in scattered coincidences. We report the results of [18F]fluoro-DOPA studies performed on six normal volunteers. Each one was scanned in the 3D and two-dimensional(2D) modes on the same tomograph. The quantitation in the 3D and 2D modes was compared for a Patlak graphical analysis with the occipital counts as the input function (Ki) and a striatooccipital ratio analysis. We find that, in 3D PET, a scatter correction is required to preserve the same quantitation as in 2D PET. When the 3D data sets are corrected for scatter, the quantitation of the [18F]fluoro-DOPA uptake, using the Patlak analysis, is similar in the 2D and 3D acquisition modes. Conversely, analysis of the striatooccipital ratio leads to higher values in 3D PET because of a better in-plane resolution. Finally, using the 3D mode, the dose injected to the subjects can be reduced by a factor greater than 1.5 without any loss in accuracy compared to the 2D mode.

6-[F-18]Fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) has been used to measure the central dopaminergic function in many species, including humans and monkeys. For transport across the blood brain barrier (BBB), FDOPA competes with plasma large neutral amino acids (LNAA). In this article we evaluate the effects of normal physiological LNAA concentration variation on BBB transport (K1) and the FDOPA uptake measurement, Ki. We also investigate a method for reducing the dependency of FDOPA quantitation on LNAA. Adult vervet monkeys ( Cercopithecus aethiops sabaeus, n = 19) were fasted overnight before FDOPA positron emission tomography scans. Blood samples were drawn for LNAA determination, metabolite analysis, and compartmental modeling. The estimated K1 and Ki were both negatively correlated with LNAA concentrations (r2= 0.51 and 0.62, respectively). Using an adjustment to K1 and Ki based on these correlations, the LNAA dependency was reduced (SD of the data for K1 was reduced by 33%, for Ki by 40%). Experiments with amino acid loading on an additional six animals indicate that BBB transport can be described using Michaelis-Menten kinetics. Results show a clear dependence of FDOPA uptake on plasma LNAA concentrations, which can be removed to increase the precision of FDOPA quantitation.

Fluorodopa (FDOPA) is an analogue of L-dihydroxyphenylalanine (L-dopa) used to assess the nigrostriatal dopamine system in vivo with positron emission tomography (PET). However, FDOPA/PET quantitation is complicated by the presence of the 3- O-methyl-FDOPA (30MFD) fraction in brain and plasma. Pretreatment with entacapone (OR-611), a peripheral catechol O-methyltransferase (COMT) inhibitor, greatly reduces the plasma 30MFD fraction and provides an ideal situation to evaluate the contribution of the plasma 30MFD fraction in several kinetic models of FDOPA uptake. We performed FDOPA/PET with and without the OR-611 preadministration in six Parkinson's disease (PD) patients. We measured the time-course of the plasma FDOPA and 30MFD fractions using high-pressure liquid chromatography (HPLC). We calculated striato-occipital ratios (SOR), and estimated the striatal FDOPA uptake rate constant graphically using the plasma FDOPA and occipital tissue time activity curves (KiFD and KiOCC, respectively). We also estimated striatal dopa decarboxylase (DDC) activity (k3D) using a model incorporating independent measurements of 30MFD transport kinetic rate constants. With the preadministration of OR-611, the pharmacological efficiency in plasma was prolonged significantly (21.1–37.7%; p < 0.01). We also observed significant mean elevations in SOR and KiOCC by 21.8 and 53.5%, respectively ( p < 0.05). KiFD and k3D did not show significant change. We conclude that OR-611 prolongs the circulation time of FDOPA in the plasma but does not alter rate constants for striatal FDOPA uptake or decarboxylation.

The analysis of positron tomographic studies of 3,4-dihydroxyphenylethylamine (dopamine) metabolism in which [18F]6-fluoro-l-3,4-dihydroxyphenylalanine (F-dopa) is used as a tracer is confounded by the presence of [18F]6-fluoro-3- O-methyl-l-3,4-dihydroxyphenylalanine (OMFD). This labeled molecule, formed by the action of peripheral cathechol- O-methyltransferase on F-dopa, crosses the blood–brain barrier and contributes to the radioactivity measured by the tomograph. Corrections for this radioactivity in the brain have been proposed. They rely upon the assumption that regional variations in the handling of this molecule by the brain are negligible. Although this assumption is pivotal for the proper quantification of dopamine metabolism using F-dopa, the distribution and kinetics of OMFD have never been studied in humans. We present results in humans that show that there is little selective regional 18F accumulation in the brain, that the distribution volume of OMFD is close to unity, and that a single, reversible compartment is adequate to model the measured time course of radioactivity after an OMFD injection. Analysis of plasma samples for labeled metabolites showed that more than 95% of the radioactivity was associated with OMFD at all times. Our results for OMFD kinetics are in accord with published results obtained in nonhuman primates and for the bidirectional transport of large neutral amino acids across the blood-brain barrier measured using a synthetic amino acid. However, our results also indicate that there are small but significant differences in OMFD kinetics in different parts of the brain that may prevent inferences about the handling of OMFD in one part of the brain from being extended to other parts of the brain.

Like native DOPA, [18F]-6-fluoro-l-3,4-dihydroxyphenylalanine ([18F]FDOPA) is subject to methylation and decarboxylation. To determine the rates of formation and elimination of [18F]FDOPA metabolites, plasma from human subjects undergoing positron emission tomographic (PET) studies was analyzed by high-performance liquid chromatography (HPLC). In addition to the principal metabolite O-methyl-[18F]FDOPA (OMe-[18F]FDOPA), two decarboxylated metabolites were detected in plasma from carbidopa pretreated subjects. The concentrations of each metabolite during 90 min following tracer injection could be described as a function of the concentration of [18F]FDOPA, and two rate constants; k0, the rate of formation, and k–1, the rate of clearance. Plasma metabolite time series generated from total plasma activity curves and measured rate constants were in close agreement with the actual concentrations determined by HPLC fractionation. Population means for k0 (0.011 ± 0.002 min−1) and k–1, (0.010 ± 0.003 min−1) were used to generate “simulated” plasma curves. The measured and generated plasma curves were used as inputs for estimation of partition and decarboxylation coefficients of [18F]FDOPA in brain. The use of generated input functions from normal population means of transfer coefficients did not introduce a systematic error into the estimate of the enzyme activity. However, the high variability of these estimates in patients precludes the use of this technique as an alterative to individual HPLC measurements.

DOPA decarboxylase is the enzyme directly responsible for the synthesis of the neurotransmitters dopamine and serotonin, and indirectly of noradrenaline, in brain. We used the decarboxylation coefficient ( kD3) of 6-[18F]fluoro-DOPA (FDOPA) to denote the relative activity of l-DOPA decarboxylase in vivo in the human brain. To determine the relative enzyme activity with positron emission tomography (PET), we evaluated the model that separates the metabolism into compartments of nondiffusible and diffusible (i.e., transient) tracer metabolites. Error analysis indicated that the least-squares optimization alone was not sufficient to yield accurate estimates of kD3 in the presence of the inherent error of PET. To improve the accuracy of the kD3 estimates by optimizing the number of parameters, we introduced biological constraints which included a tracer partition volume ( Ve) common to frontal cortex and striatum, and a fixed ratio ( q) between the blood–brain barrier transport coefficients of O-methyl-[18F]fluoro-DOPA and FDOPA, the two sources of radioactivity in plasma. We found that a two-step analysis yielded sufficiently accurate estimates of kD3. The two steps include the initial estimation of the partition volume in frontal cortex and the subsequent use of this value to determine kD3 in striatum and other structures. We studied twelve healthy controls (age 45 ± 15 years). The average kD3 value was 0.081 ± 0.024 min−1 (coefficient of variation (COV) 30%) for caudate nucleus, 0.074 ± 0.013 min“1 (COV 18%) for putamen, and 0.010 ± 0.005 min−1 (COV 50%) for cerebral cortex.

This article describes a simplified method for the determination of the l-[18F]6-fluorodopa (FDOPA) fraction time course that takes advantage of the strong correlation between the radioactivity ratio (metabolites/FDOPA) and time. Serial arterial blood samples are collected for assay of plasma total radioactivities following an intravenous injection of FDOPA into carbidopa-pretreated subjects. In addition, a single plasma sample, collected late in the study and analyzed for FDOPA fraction, is sufficient to determine accurately the time course of the FDOPA concentration in plasma. The validated straight-line method greatly simplifies blood analysis for routine positron emission tomography FDOPA studies.

Kinetics of l-3,4-dihydroxy-6-[18F]fluorophenylalanine (FDOPA) in striatum and cerebellum were measured in 10 normal human subjects with positron emission tomography (PET) from 0 to 120 min after an intravenous bolus injection of the tracer. The time course of the arterial plasma concentrations of the tracer and its metabolites was also assayed biochemically. FDOPA compartmental models that are based on biochemical information were investigated for their consistency with the measured striatal and cerebellar tissue kinetics. A modeling approach was also developed for separating plasma FDOPA and metabolite time-activity curves from the measured total 18F time-activity curve in plasma. Results showed that a model consisting of three separate compartments for tissue FDOPA, tissue 6-[18F]fluorodopamine (FDA) and its metabolites, and tissue l-3,4-dihydroxy-6-[18F]fluoro-3- O-methylphenylalanine (3-OMFD) could describe adequately the striatal kinetics in humans. Based on this model, the FDOPA transport constant across the blood–brain barrier (BBB) ( K1), the FDOPA decarboxylation rate constant ( k3), and the turnover rate constant of FDA and its metabolites ( k4) could be estimated by model fitting to the tissue kinetics and were found for the normal subjects to be 0.031 ± 0.006 ml/min/g (mean ± SD), 0.041 ± 0.015/min, and 0.004 ± 0.002/min, respectively. About 50% of the FDOPA that crossed the BBB from plasma to striatum was decarboxylated. The decarboxylation constant with respect to plasma FDOPA ( K3) was 0.015 ± 0.003 ml/min/g. The BBB transport corresponded to a permeability–surface area product of 0.032 ml/min/g for FDOPA. For 3-OMFD, the BBB transport was 1.7 times faster. The effects of tissue heterogeneity on the FDOPA kinetics and on the estimated model parameters were also investigated. The usefulness and implications of these findings for interpretation of PET FDOPA studies are discussed.

Characterization of peripheral and cerebral l-3,4-dihydroxy-6-[18F]fluorophenylalanine (FDOPA) metabolism in humans and monkeys has shown FDOPA to be an analogue of l-DOPA for the study of the dopaminergic system with positron emission tomography (PET). In human studies with carbidopa pretreatment, l-3,4-dihydroxy-6-[18F]fluoro-3- O-methylphenylalanine (3-OMFD) was the only FDOPA metabolite detected in plasma. FDOPA administration in monkeys resulted in selective accumulation of FDOPA metabolites in central dopaminergic regions, whereas 3-OMFD of peripheral origin was uniformly distributed among putamen, caudate, frontal cortex, and cerebellum. At 60 min, 3-OMFD and 6-[18F]fluorodopamine (FDA) each represented ∼35% of the total activity, the remainder being FDOPA and FDA metabolites. These data on monkey and human FDOPA metabolism provide the basis for the configuration of an FDOPA tracer kinetic model with PET.

In a study designed to reveal the rates of blood–brain transfer and decarboxylation of fluoro-L-3,4-dihydroxyphenylalanine (FDOPA), we discovered a major discrepancy between the DOPA decarboxylase activity reported in the literature and the rate of FDOPA decarboxylation measured in the study. “Donor” rats received intravenous injections of 6 mCi fluorine-18-labeled FDOPA. The donor rats synthesized methyl-FDOPA. Arterial plasma, containing both FDOPA and methyl-FDOPA, was sampled from the donor rats at different times and reinjected into “recipient” rats in which it circulated for 20 s. The blood–brain clearance of the mixture of labeled tracers in the plasma was determined by an integral method. The individual permeabilities were determined by linear regression analysis, according to which the average methyl-FDOPA permeability in the blood–brain barrier was twice that of FDOPA, which averaged 0.037 ml g−1 min−1. The permeability ratio was used to determine the fractional clearance from the brain of FDOPA (and hence of methyl-FDOPA), which averaged 0.081 min−1. In the striatum, the measured average FDOPA decarboxylation rate constant ( kD3) was 0.010 min−1, or no more than 1% of the rate of striatal decarboxylation of DOPA measured in vitro and in vivo. We interpreted this finding as further evidence in favor of the hypothesis that striatum has two dopamine (DA) pools, of which only DA in the large pool is protected from metabolism. Hence, no more than 1% of the quantity of fluoro-DA theoretically synthesized was actually retained in striatum.

The effects of elevated circulating concentrations of prolactin were examined on neurochemical estimates of activity of dopaminergic neurons in the infundibulum/pituitary stalk of Holstein bull calves (8-10 weeks of age). Activity of these neurons was estimated by measuring the accumulation of dihydroxyphenylalanine, the immediate precursor of dopamine, 15 min after an intravenous injection of the aromatic L-amino acid decarboxylase inhibitor, 3-hydroxybenzylhydrazine. Subcutaneous injections of the dopamine antagonist haloperidol every 6 hr for 1 day increased serum concentrations of prolactin and accumulation of dihydroxyphenylalanine in the infundibulum/stalk. Intravenous infusions of prolactin for 1 or 9 days increased accumulation of dihydroxyphenylalanine in the infundibulum/stalk, indicating that these neurons remain responsive to elevated prolactin for at least 9 days. It is concluded that elevated concentrations of prolactin in blood stimulate dopaminergic neurons in the infundibulum/pituitary stalk of bull calves. We speculate that these neurons may be analogous to the tuberoinfundibular dopaminergic neurons that regulate prolactin in rats.

A qualitative gas chromatographic analysis of trimethylsilylated ethyl acetate extracts of melanotic urine revealed 5 indolic compounds, which have been identified as substituted 5,6-dihydroxyindoles. Ethyl acetate extracts of melanotic urines at pH 2.0 contained isomeric 5-hydroxy-6-methoxy and 6-hydroxy-5-methoxy-indolyl-2-carboxylic acids which were not separable under the conditions used. A careful hydrolysis of melanotic urine with a Helix pomatia preparation followed by extraction at pH 6.5 in a nitrogen atmosphere released 3 additional indolic compounds from their conjugated form. Using gas chromatographic-mass spectrometric analysis they were identified as 5-hydroxy-6-methoxy, 6-hydroxy-5-methoxyindole and 5,6-dihydroxyindole.

SummaryThe concentrations of liver free amino acids in l-Dopa-treated and control rats were assayed by a Jeol amino acid analyzer. Subcutaneous injections of 500 mg/kg l-Dopa daily for 8 days incre...

This study investigated shorter and longer range effects of L-Dopa therapy in parkinsonism on critical flicker frequency (CFF) scores. Three equated groups of 15 Ss were selected to include “short-term,” “long-term,” L-Dopa patients and a control group. Binocular CFF thresholds were obtained for each S on two separate occasions. Results indicate that: (1) the control group scored significantly higher on CFF indicating superior neural integration when compared with the “short-term” or “long-term” L-Dopa group; (2) the “short-term” L-Dopa group scored significantly higher than the “long-term” L-Dopa group demonstrating better cerebral efficiency. Evidence suggests that a peculiar clinical state interfering with neural transmission may develop in parkinsonian patients on L-Dopa therapy prolonged 2 yr. or more.

In the melanocyte, tyrosinase is known as the dey enzyme for melanin formation. Purified tyrosinase protein was prepared that was capable of oxidizing tyrosine. The localization of tyrosinase antigen in the melanocyte was studied using antiserum against tyrosinase. DOPA (L-3,4-dihydroxyphenylalanine)-reaction product and tyrosinase antigen were found on the same organelles i.e., premelanosomes, melanosomes, GERL, and Golgi vesicles. This result seems to suggest that it is cytochemically appropriate to use DOPA as the substrate of tyrosinase. It appeared that tyrosinase antigen was present as granule-like structures inside GERL cisterna and associated with its membrane.

Summaryl-3-Methoxytyrosine did not substitute for l-dopa, in increasing the threshold for electrically induced extensor seizures in mice, or in restoring the ability of d-methamphetamine to stimulate motor activity in rats treated with dl-α-methyltyrosine. At high doses l-3-methoxytyrosine inhibited the effect of l-dopa in both tests, l-3-Methoxytyrosine partially inhibited the increase in brain levels of dopa, dopamine, and norepinephrine produced by l-dopa. It is suggested that l-3-methoxy tyrosine may interfere with the uptake of l-dopa from the peritoneal cavity and from the blood into the brain.

Copper chelation of nitrosotyrosine has been found useful for histochemical localization for tyrosine in thick, fresh sections of large celled plant tissues. The nitrous acid reaction for ortho-dihydroxyphenolics also has been found useful for localization of chlorogenic acid, caffeic acid and dihydroxyphenylalanine in plant tissues. Application of these tests separately to serially adjacent sections demonstrated the distribution of tyrosine and chlorogenic acid in different plant tissues. Tests tube reactions on known substances verified specificity and also demonstrated that the presence of other amino acids and phenolics did not interfere with the positive test for tyrosine. The color reactions are sufficiently intense for stereoscopic microscopy and tested sections may be measured photometrically. Further adaptability of the nitrosotyrosine-metal chelate reaction to procedures for ultrastructural localization is suggested.

The catecholamines—dopa, dopamine, adrenaline and noradrenaline—are known to produce fluorescing 3,4-dihydroisoquinoline derivatives on condensation with formaldehyde. These dihydro products could be converted to 1,2,3,4-tetrahydroisoquinoline derivatives by borohydride reduction and in turn again to dihydro forms by re-exposing to formaldehyde vapor. Periodic acid was found to cleave the —C—C—bond at 3—4 position of tetrahydroisoquinoline derivative of noradrenaline only. This prevented the cyclitization of heterocyclic ring of tetrahydro form to give fluorescing dihydro product. This reaction can be utilized for the localization of noradrenaline in histochemical fluorescent preparations. Noradrenaline is prevented from regaining its fluorescence on re-exposure to formaldehyde by imposing periodic acid oxidation after borohydride bleaching.

SummaryThe effect of L-dopa and isoproterenol in DNA, RNA, and protein synthesis was measured in the salivary glands, brain, and liver of the mouse. These data indicate that L-dopa does not cause gross changes in nucleic acid and protein synthesis.We thank Dr. George C. Cotzias for stimulating this work and Miss Roberta Klimaski for technical assistance.

SummaryIn these experiments rats are reserpinized, and it is demonstrated that α-methyldopa reverses the tyramine pressor response blockade by reserpine. Diethyldithiocarbamic acid will inhibit the reversal process by a-methyldopa- a-methyldopamine, dopamine, and dopa. Disulfiram inhibits the reversal process of a-methyldopa and dopa. Disulfiram or diethyldithiocarbamic acid alone will not inhibit the tyramine response. Diethyldithiocarbamic acid is most potent of the two agents used in inhibiting the reversal process.Addendum: Musacchio, J. M., Bhagat, B., Jackson, C. J. and Kopin, I. J., J. Pharmacol., 1966 vl52, 293, have used nearly the same technique to show inhibition by disulfiram of the restoration by dopamine and α-methyldopamine of the response to tyramine in reserpine-treated cats.

SummaryAcute administration of l-dopa (500-750 mg) lowered plasma TSH concentration and depressed an increase of plasma TSH in response to TRH in patients with primary hypothyroidism. In contrast, chronic administration of small or large doses of l-dopa failed to depress the development of goiter in rats treated with methimazole, indicating that plasma TSH concentration was not lowered by l-dopa in methimazole fed animals. Acute administration of l-dopa also failed to prevent an increase of intrathyroidal colloid droplets produced by an increase of TSH secretion in response to TRH. It is suggested that there is species specificity in the TSH response to the administration of l-dopa.

Over a two-year period one hundred and sixty-two patients with Parkinson's disease have been treated with L-DOPA in a cooperative study involving four hospitals in the Montreal area. The clinical results for the first consecutive one hundred patients treated for longer than three months are reviewed. These observations warrant the conclusion that L-DOPA is by far the best currently available treatment for Parkinson's disease. Some potentially troublesome side effects still exist and these require further investigations.

The known capacity of certain cells in the rodent intestine to convert dopa to melanin has been investigated. The cells become pigmented following incubation in dopa solutions at pH 6.8 for 24 hr, but not when incubated in tyrosine. The reaction is prevented by pretreatment with buffer solution at 100°C and ethylene glycol, but not by buffer at 60°C, ethanol or benzene. The reaction is inhibited by sodium azide, ascorbic acid and catalase, but not by protamine sulphate, 4-chlororesorcinol and sodium diethyldithiocarbamate. These results suggest that the enzyme concerned is a peroxidase. The cells concerned are confined to the lamina propria mucosae, and are found in the connective tissue of the spleen and liver. They are also present in peripheral blood. They are eosinophilic and have an affinity for pyronin. They do not contain chromaffin material, nor do they stain with nuclear fast red. It is concluded that they are granular leucocytes, probably eosinophil granulocytes.

An attempt is made to provide a cohesive conceptual frame upon which may be placed the recent advances in our understanding of the psychobiology of the affective disorders. Major literature in the areas of neurophysiology, endocrinology and biochemistry is reviewed and placed within a dynamic model which draws heavily upon ideas previously put forward by Meyer and von Bertalanffy. Phenomenologic and semantic difficulties which have retarded research in the depressions are also addressed.

1. It is demonstrated that the interpretation of the histochemical dopa reaction, discovered by Bloch, correlated with mammalian melanogenesis in vivo has been greatly influenced by the analogy of the tryosinase-tyrosin reaction in vitro with the same process. 2. Repeating Bloch's histochemical pretreatment experiments on fixed tissue proved that the extreme lability found by Bloch must be attributed to detachment of the factor from the section or insufficient control of pH of the pretreatment solution. 3. Sufficient lability remains to be in line with an enzymatic nature, though classifying the factor in the realm of copper containing enzymes must be considered premature for lack of comparable data in the extract enzymological field.

Condensation with glyoxylic acid vapor, recently introduced as a highly sensitive method for fluorescence histochemical visualization of biogenic monamines, has been found to allow also the demonstration of certain tryptophan- or dopa-containing peptides. Thus, in model experiments, treatment with glyoxylic acid vapor induced fluorescence from di- and tetrapeptides with tryptophan or dopa in NH2-terminal or COOH-terminal position. Peptides without tryptophan or dopa in terminal positions gave no observable fluorescence. Differences were recorded between peptides with NH2-terminal and COOH-terminal tryptophan with respect to their fluorescence yields under different reaction conditions and the spectral characteristics of the fluorophores (excitation/emission maxima: 375-380/500-520 nm and 340-370/435 nm, respectively). In addition, the tryptophan-containing peptides could be distinguished microspectrofluorometrically from the dopa-containing peptides. Thus, the fluorophore of the NH2-terminal dopa peptide had excitation/emission maxima at 330 and 380/495 nm, and the corresponding values for the fluorophore of the COOH-terminal dopa peptide were 330 and 370/460 nm. The glyoxylic acid treatment produced intense fluorescence in certain endocrine cell systems in pituitary, thyroid and gastric mucosa, previously suggested to store peptides with NH2-terminal tryptophan. The present findings support this hypothesis.

S~mnary.-1 l patients undergoing L-dopa therapy for Parkinson's disease were evaluated using 3 psychomotor measures designed to assess functioning of the upper extremity. The patients were tested prior to L-dopa therapy, when they had reached optimum dosages of L-dopa, and finally, during a period when they had been placed on a placebo. Significant differences were found on two of the three measures between pretreatment and L-dopa periods, and between pretreatment and placebo periods. No significant differences were found between L-dopa and placebo periods, however. The recent use of levodopa (L-dopa) in the management of Parkinson's disease has produced a number of studies which attest to the efficacy of this drug (Barbeau, 1961; Birkmayer & Hornykiewicz, 1961; Cotzias, et al., 1967; Yahr, et al., 1968; Cotzias, et al., 1969). This study is an attempt to measure change objectively and to evaluate three different tests of certain aspects of upper extremity motor performance ( 1 ) prior to the administration of L-dopa, (2) at a point when the patient is considered to be on optimum treatment dosages of Ldopa, and (3) following a period during which the patient has been on placebo. Ss were 11 patients out of an original group of 16 who were the initial group of patients at the Mayo Clinic to receive L-dopa therapy. Two of the remaining five patients were not tested because their mental or physical status prevented them from comprehending or executing the particular tasks; three other patients were excluded from the analysis because their data were incornplete. All patients had the diagnosis of Parkinson's disease and had been so diagnosed for varying lengths of time ranging from 2 to 30 yr. (average, 5.6 yr ) They ranged in age from 40 to 77 yr. (average, 58.3 yr.) . Procedare

SummaryColorimetrie assay of a large number of human and animal brains for “encephalin” revealed a rather uniform pattern of intracerebral distribution in various species. There was no appreciable difference between the encephalin content and its distribution in the brains of “normal” and “psychotic” humans. No major changes were observed under the influence of various hormones, adrenalectomy, hypophysectomy, convulsive drugs, electrical stimulation of the brain, narcotics, conditions of stress, acetylcholine, and X-free diet. Only the injection of dihydroxyphenylalanine was followed by a very marked temporary increase of the colorimetric readings, which suggests either a selective absorption of this substance by the brain or its conversion into encephalin.

A group of apparently unrelated endocrine cells, some in endocrine glands, others in nonendocrine tissues, share a number of cytochemical and ultrastructural characteristics. These characteristics, from the initial letters of four of which the term APUD is derived, indicate the possession of a common metabolic pattern and common synthetic, storage and secretion mechanisms. It is postulated that the various characteristics reflect the production and storage of hormone precursor protein which has a predominantly random coil conformation. Several explanations are possible to account for the APUD characteristics but if the cells, in fact, share a common ancestor the only possible candidate is the neural crest cell.