Dopamine hydroxylase assay

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. 

Dopamine /3-hydroxylase is a monoxygenase that catalyzes the hydroxylation of dopamine to form norepinephrine. This enzyme is localized in the chromaffin granules of the adrenal medulla and in the storage vesicles of central and peripheral catecholaminergic neurons. Since these compounds are unstable, this activity is often assayed by following the formation of octopamine from tyramine. For example, in the assay developed by Feilchenfeld et al. (1982), the reactant tyramine was separated from the product octopamine by reversed-phase, ion-paired HPLC (/uBondapak C18 using a mobile phase of 17% (v/v)... 

An important aspect of the preparation and isolation of subcellular particles from brain regions is the criteria by which purity is assessed. Electron microscopy of the various subcellular fractions can provide among the best pieces of evidence for the presence in the preparation of the organelles or subcellular fragments of interest. However, a number of biochemical markers (usually enzymes) that have been established to be present in certain fractions can also be assayed to demonstrate the enrichment of the organelle of interest. For instance, acetylcholinesterase is a common marker for synap-tosomes dopamine-P-hydroxylase is a marker for catecholamine storage vesicles within the synaptosome and cytochrome c oxidase is a marker for mitochondria. Most of the enzymatic markers can be assayed routinely. 

Assay of phenylalanine hydroxylase in a liver biopsy from one patient showed 20% of normal adult control values, but dihydropteridine reductase activity (Fig. 20.2) was less than 1% of normal in the liver, brain, and other tissues. This latter deficiency presents regeneration of tetrahydrobiopterin, the cofactor for the hydroxylase reaction. Since the reductase enzyme reaction regenerates the cofactor for tyrosine and tryptophan hydroxylase, catecholamine and serotonin synthesis are compromised as well. Patient studies are scanty, but in one patient dopamine and serotonin were decreased in the cerebrospinal fluid, brain, and various other tissues, while norepinephrine metabolites were normal. While phenylalanine hydroxylase activity was lower than that of adult controls, it was not determined whether this value represented significantly decreased activity in children. 

Electrochemical detection also offers an alternative approach to amine enzyme activity measurement as the assays for the precursors, the amines, and their metabolites are adequately sensitive. Modifications to the normal assay are required especially if the substrate, which will be added in excess, is electroactive and separated on the same LC column. For example, dopamine-p-hydroxylase activity is measured by incubating dopamine with the required cofactors for a fixed time and then measuring the amount of norepinephrine formed (Davis and Kissinger, 1979). However, sub-... 

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