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Dopaminergic neuronal cell degeneration is the principal characteristic feature of the neuropathology of Parkinson disease. Cultures of mesencephalic neurons are widely used as a source of dopaminergic neurons for the study of mechanisms implicated in dopaminergic cell death and for the evaluation of potential dopaminergic neuroprotective agents, including neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat mesencephalic cell cultures and their application to evaluating the effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium and the neuroprotective action of brain-derived neurotrophic factor.

Recently many studies have focused on long-term follow-up of phenylketonuria (PKU McKusick 261600) patients, especially regarding discontinuation of diet in adulthood and subsequent possible neurological involvement. Neurological symptoms have been observed in some patients after discontinuation of diet, with improvements after reintroduction of a phenylalanine-restricted diet (Pietz et al 1998). Brain magnetic resonance imaging (MRI) revealed white-matter abnormalities in patients with neurological impairment but also in many patients without neurological symptoms (Burgard et al 1999). Myelin changes observed by brain MRI are similar in early-treated, late-treated and untreated patients, differing only in extension. Neurological investigations of treated adult PKU patients revealed minor neurological signs (tremor, brisk deep tendon reflexes, clumsy motor coordination) indicating the possibility of a specific neurological syndrome in adult PKU patients off diet. Even early-treated phenylketonuric patients show subtle symptoms of CNS damage, in addition to neuropsychological, neurophysiological and brain imaging abnormalities (Pietz et al 1998). Therefore, further investigations are necessary to evaluate the presence of biochemical changes, even in early-treated patients, that could be implicated in the neurological alterations. The aim of our study was to investigate dopamine and serotonin metabolism (homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), respectively) in early-treated PKU patients with white-matter abnormalities on magnetic resonance imaging.

N-Acetylglutamate synthetase (NAGS, EC 2.3.1.1), the deficiency of which (McKusick 23735) in humans has only previously been reported three times (Bachmann et al 1981, 1988; Elpeleg et al 1990), is a mitochondrial enzyme that normally catalyses the synthesis of N-acetylglutamate (NAG), an obligate activator of carbamoyl phosphate synthetase (CPS), which initiates the first step of urea synthesis from ammonia

The allopurinol challenge test was performed on 44 healthy subjects (28 children and 16 adolescents) in order to establish normal values of urinary orotic acid excretion following allopurinol ingestion in the paediatric population. The subjects were divided into three groups according to their age: 6 months to 6 years; 6 years to 10 years; and 10 years to 17 years. They were given 100 mg, 200 mg, or 300 mg of allopurinol, respectively (based on age) in a single oral dose. Maximum peak urinary orotic acid levels following ingestion of allopurinol were 13.0 (n = 14), 9.3 (n = 14), and 10.2 (n = 16) mumol/mmol creatinine in the three groups, respectively. In all children tested the peak orotic acid level was 3.1 +/- 2.7 mumol/mmol creatinine (mean +/- SD, n = 44). This allopurinol challenge test was also performed in six children with urea-cycle disorders, including five females with ornithine transcarbamylase (OTC) deficiency, all of whom demonstrated abnormally elevated levels of urinary orotic acid (peak levels of 26-134 mumol/mmol creatinine) following allopurinol ingestion.

we describe a new RP-HPLC assay to measure orotate concentration in urine. The method is rapid, sensitive and precise. It is particularly suitable for the diagnosis of inherited metabolic diseases and deranged orotate metabolism.