Dopamine assay

K14. Koslow, S. H., Cattabeni, F., and Costa, E., Norepinephrine and dopamine assay by mass fragmentography in the picomole range. Science 176, 177-180 (1972). 

A procedure for the fluorimetric determination of 3-methoxytyra-mine was described by Carlsson and Waldeck in 1964.304 This procedure, which resembled the dopamine assay procedures previously described (cf. Section V, E, 3), involved the oxidation of the amine with iodine, presumably to an aminochrome (cf. the oxidation of meta-nephrine (Section II, D, 1)] which was subsequently rearranged to a fluorescent derivative.304... 

It is appropriate at this juncture to illustrate the power of chemiluminescence in an analytical assay by comparing the limits of sensitivity of the fluorescence-based and the chemllumlnescence-based detection for analytes in a biological matrix. The quantitation of norepinephrine and dopamine in urine samples will serve as an illustrative example. Dopamine, norepinephrine, and 3,4-dihydroxybenzy-lamine (an internal standard) were derivatized with NDA/CN, and chemiluminescence was used to monitor the chromatography and determine a calibration curve (Figure 15). The limits of detection were determined to be less than 1 fmol injected. A typical chromatogram is shown in Figure 16. 

Biochemical analyses of 6-OHDA-injected animals revealed a 93 percent depletion of dopamine. The tissue was assayed using electrochemical detection following separation by high-pressure liquid chromatography (Felice et al. 1978). recorded as ng/mg protein in the nucleus accumbens and compared to control rats with sham lesions (sham=65.5 4.4, lesion=4.9 1.5 t(39)=23.4). A lesion was defined as complete if 75 percent or more of the dopamine was determined to be depleted from the nucleus accumbens compared to mean sham group values. 

Figure 4. DDC (A), serotonin (B), and tyrosine hydroxylase (C) immunore-activity in the posterior region of a wild-type Drosophila ventral ganglion. Tyrosine hydroxylase (TH) encodes the rate-limiting step in dopamine biosynthesis and is a marker for dopamine cells. B and C are the same CNS assayed for both serotonin and TH. M, medial dopamine neurons VL, ventrolateral serotonin neurons DL, dorsolateral dopamine neurons. Short unmarked arrows in C show vacuolated cells that do not contain DDC immunoreactivity. The immunoreactivity in these cells may represent a nonspecific cross-reactivity of the rat TH antibody. The length bar in A is 50 pM. The images are confocal projections generated on a Molecular Dynamics-2000 confocal laser scanning microscope.

Patients sustain convulsions and neurological deterioration. The urine contains low levels of the metabolites of serotonin, norepinephrine and dopamine. The reductase also plays a role in the maintenance of tetrahydrofolate levels in brain, and some patients have had low folate levels in the serum and CNS. Treatment has been attempted with tryptophan and carbidopa to improve serotonin homeostasis and with folinic acid to replete diminished stores of reduced folic acid. This therapy is sometimes effective. Diagnosis involves assay of DHPR in skin fibroblasts or amniotic cells. Phenylalanine hydroxylase activity is normal. 

Finally, loop regions have also been implicated in ligand interactions or their modulation. Chimeras generated between NET and DAT were assayed for ethanol sensitivity and revealed that Glyl302.68 and Ilel373.3i in IL1 are important for ethanol modulation of DAT activity (78). In addition, mutation to alanine of Prol363 30 in IL1, as well as Pro553i2.43 in EL6, decreased the apparent affinity for dopamine uptake (59). 

Slow-onset, long duration dopamine reuptake inhibitors with reduced potential for substance abuse have been suggested as therapies for psychostimulant addiction [33-35]. A series of slow-onset, long duration N-alkyl analogues of methylphenidate were recently reported to have enhanced selectivity for the dopamine transporter [34]. A representative compound is 13, an RR/SS diastereomer (DAT K, = 16nM, SERT K = 5900 nM, NET K-, = 840 nM). In a locomotor activity assay in mice, 13 has a slow onset of activity (20-30 min) with peak activity occurring between 90 and 120 min. In contrast, both methylphenidate and cocaine are active within 10 min and reach peak activity within 30 min. 

Biogenic amines are of great interest to researchers because of their potential roles in several psychiatric and neurological disorders. They include dopamine (DA), noradrenaline (NA), 5-hydroxytryptamine (5-HT, serotonin), histamine, and trace amines such as 2-phenylethylamine (PEA), tyramine, octopamine, phenylethanolamine, and tryptamine (Coutts and Baker, 1982). Although GC assays for DA, NA, and 5-HT are available, HPLC analysis with electrochemical detection has for many years now been the method of choice for analysis of these neurotransmitter amines. 

The use of HPLC to analyze biogenic amines and their acid metabolites is well documented. HPLC assays for classical biogenic amines such as norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin) and their acid metabolites are based on several physicochemical properties that include a catechol moiety (aryl 1,2-dihydroxy), basicity, easily oxidized nature, and/or native fluorescence characteristics (Anderson, 1985). Based on these characteristics, various types of detector systems can be employed to assay low concentrations of these analytes in various matrices such as plasma, urine, cerebrospinal fluid (CSE), tissue, and dialysate. 

Lakshmana MK, Trichur TR. 1997. An isocratic assay for norepinephrine, dopamine and 5-hydroxytryptamine using their native fluorescence by high-performance liquid chromatography with fluorescence detection in discrete brain areas of rat. Anal Biochem 246 166-170. 

Hows ME, Lacroix L, Heidbreder C, Organ AJ, Shah AJ. 2004. High-performance liquid chromatography/tandem mass spectrometric assay for the simultaneous measurement of dopamine, norepinephrine, 5-hydroxytryptamine and cocaine in biological samples. J Neurosci Methods 138 123. Hows ME, Organ AJ, Murray S, Dawson LA, Foxton R, et al. 

Techniques that measure mRNA expression and ligand binding assays that measure receptor expression have been used extensively to identify those nAChR subtypes that are expressed in dopamine neurons. In situ hybridization studies using mouse (Marks et al. 1992 Grady et al. 1997) and rat (Le Novere et al. 1996) brain have detected the mRNAs for all of the known nAChR subunits, except a2 and p4, in... 

Besides the poor specificity of many of the assays used to determine plasma drug concentrations, another problem which has arisen from these studies has been the length of the "wash-out" period necessary before the patient is given the neuroleptic under investigation. As a result of the prolonged duration of blockade of dopamine receptors in the brain by conventional neuroleptics and their metabolites, it is necessary to allow a wash-out period of several weeks before the patients are subject to a pharmacokinetic study. This raises serious ethical questions. Perhaps with the advent of new imaging techniques it may be possible in the near future actually to determine the rate of disappearance of neuroleptics from the brain of the patient. This may enable the relationship between plasma concentration and clinical response to be accurately determined. 

Fig. 7.4 Structures of compounds used in competitive MS binding assays for dopamine Di and D2 receptors.

Fig. 7.5 Schematic flowchart of the competitive MS-binding assay quantifying the nonbound marker employed for dopamine Di receptors. After incubation of the target (Di receptor) in presence of the marker (SCH 23390) and a test compound, the binding samples are centrifuged to separate bound from nonbound marker. The nonbound marker in the resulting supernatant is quantified by LC-ESI-MS/MS without further sample preparation.

Fig. 7.6 Nonbound SCH 23390 in a competitive MS binding assay for dopamine Di receptors monitored at a transition from 288.1 91.2 m/z from binding samples without or with (+)-butaclamol. Intensity (/) is shown (a) without (+)-butaclamol, (b) with 30 nM (+)-butaclamol, (c) with 10 pM (+)-butaclamol. (a-c) Representative chromatograms after HPLC separation (RP8 column solvent CH3CN/0.1% HCOOH in H2O 1 1 300 pL min ).

Fig. 7.7 Representative binding curve obtained by nonlinear regression from a competitive MS binding assay for dopamine Di receptors, in which (+)-butaclamol competes with SCH 23390 as marker. The points describe nonbound SCH 23390 quantified by LC-ESI-MS/MS. Data reflect means (+s) from binding samples, each performed in quadruplicate.

Table 7.1 Affinities (mean + SEM, n = 3) for dopamine antagonists at D] receptors obtained by MS-binding assays and radioligand binding assays, respectively [61],...

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