Aida Ormazabal

HPLC with electrochemical and fluorescence detection procedures for the diagnosis of inborn errors of biogenic amines and pterins

The analysis of biogenic amines (BA) and pterins in cerebrospinal fluid (CSF) is essential for the early diagnosis of neurotransmission defects in the paediatric age. Our aim was to standardize previously reported HPLC procedures for the analysis of BA and pterins in CSF and to establish reference values for a paediatric population. Samples from 127 subjects (age range 11 days to 16 years; average 3.8) were analyzed by HPLC with electrochemical and fluorescence detection. Both BA (homovanilic and 5-hydroxyindoleacetic acid) and pterins (neopterin and biopterin) concentrations in CSF showed a negative correlation with age. This finding led us to stratify reference values into six groups according to age. In conclusion, analysis of BA and pterins in CSF by HPLC procedures is a useful set of tools for the diagnosis of inborn errors of metabolism of these compounds. The establishment of reference intervals may be difficult, since there is a strong correlation between BA concentrations and the age of controls and, as a result, a large number of CSF samples from control populations would be necessary for this purpose.

Cerebral folate deficiency and leukoencephalopathy caused by a mitochondrial DNA deletion.

Our aim was to describe a child with an incomplete form of Kearns–Sayre syndrome who presented profound cerebrospinal fluid (CSF) folate deficiency and his response to folinic acid supplementation

Mitochondrial diseases associated with cerebral folate deficiency

Cerebral folate deficiency (CFD) has been defined as any neuropsychiatric condition associated with isolated lowering of 5-methyltetrahydrofolate (5-MTHF) levels in CSF and normal systemic folate metabolism.1 CFD has been detected in the infantile-onset CFD syndrome (mediated by serum folate receptor [FR] autoantibodies of the blocking type1) and Aicardi-Goutieres and Rett syndromes. In Kearns-Sayre syndrome (KSS), systemic folate deficiency or low CSF 5-MTHF have long been recognized.2,3 Because active transport of 5-MTHF across the choroid plexus epithelial cells is mediated by ATP-dependent processes, we conducted a study to determine CSF 5-MTHF in a series of patients with mitochondrial disorders. ### Methods. Twenty-eight patients with different mitochondrial disorders and fulfilling the previously defined diagnostic criteria4 were recruited from the Hospital Sant Joan de Deu, Barcelona, Spain (21 patients), the University Clinic Aachen, Germany (5 patients), and Hospital 12 de Octubre, Madrid, Spain (2 patients). Diagnosis included KSS (5 patients), neuropathy, ataxia, and retinitis pigmentosa (1 patient), mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (1 patient), and various clinically heterogeneous respiratory chain enzyme deficiencies (RCD) (21 patients). Brain MRI was performed for each patient. Serum folate and CSF 5-MTHF, glucose, protein, and cells were determined as per established protocol.5 CSF 5-MTHF …

Kearns-Sayre syndrome: cerebral folate deficiency, MRI findings and new cerebrospinal fluid biochemical features.

We evaluated cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF), biogenic amines, and white matter status in six Kearns-Sayre syndrome (KSS) patients. They presented severe 5-MTHF deficiency. A significant negative correlation was observed between CSF 5-MTHF and protein concentration. CSF homovanillic acid was clearly high. Regarding neuroimaging, the main feature was hyperintensity in the basal ganglia, brainstem, and cerebral/cerebellar white matter. The severity of hemispheric white matter disturbances appeared to be qualitatively associated with 5-MTHF values. The negative correlation between 5-MTHF and proteins supports the hypothesis of impaired choroid plexus function. Interestingly, despite very low 5-MTHF, clearly high neurotransmitter metabolites were found.

Evaluation of CSF neurotransmitters and folate in 25 patients with Rett disorder and effects of treatment

BACKGROUND Rett disorder (RD) is a progressive neurodevelopmental entity caused by mutations in the MECP2 gene. It has been postulated that there are alterations in the levels of certain neurotransmitters and folate in the pathogenesis of this disease. Here we re-evaluated this hypothesis. PATIENTS AND METHODS We evaluated CSF folate, biogenic amines and pterines in 25 RD patients. Treatment with oral folinic acid was started in those cases with low folate. Patients were clinically evaluated and videotaped up to 6 months after therapy. RESULTS CSF folate was below the reference values in 32% of the patients. Six months after treatment no clinical improvement was observed. Three of the four patients with the R294X mutation had increased levels of a dopamine metabolite associated to a particular phenotype. Three patients had low levels of a serotonin metabolite. Two of them were treated with fluoxetine and one showed clinical improvement. No association was observed between CSF folate and these metabolites, after adjusting for the patients age and neopterin levels. CONCLUSION Our results support that folinic acid supplementation has no significant effects on the course of the disease. We report discrete and novel neurotransmitter abnormalities that may contribute to the pathogenesis of RD highlighting the need for further studies on CSF neurotransmitters in clinically and genetically well characterized patients.

Mitochondrial diseases mimicking neurotransmitter defects

OBJECTIVES Mitochondrial disorders are clinically heterogeneous. We aimed to describe 5 patients who presented with a clinical picture suggestive of primary neurotransmitter defects but who finally fulfilled diagnostic criteria for mitochondrial disease. METHODS We report detailed clinical features, brain magnetic resonance findings and biochemical studies, including cerebrospinal fluid (CSF) biogenic amine and pterin measurements, respiratory chain enzyme activity, and molecular studies. RESULTS The 5 patients had a very early onset age (from 1 day to 3 months) and a severe clinical course. They all showed a clinical picture suggestive of infantile hypokinetic-rigid syndrome (hypokinesia, hypomimia, slowness of reactions, tremor), other abnormal movements (myoclonus, dystonia), axial hypotonia, limb hypertonia, feeding difficulties, and psychomotor delay. Abnormal CSF findings among the 4 patients without treatment included low levels of homovanillic acid (HVA) in 3 patients, with associated low 5-hydroxyindoleacetic acid (5-HIAA) concentrations in two of them. Absent or mild and transitory improvement was observed after treatment with L-dopa. A diagnosis of mitochondrial disorder was finally made due to the appearance of hyperlactacidemia, diverse respiratory chain defects, and multisystemic involvement. CONCLUSIONS Secondary neurotransmitter disturbances may occur in mitochondrial diseases. Differential diagnosis of hypokinetic-rigid syndrome presenting in infancy could also include paediatric mitochondrial disorders.

Levodopa‐induced dyskinesias in tyrosine hydroxylase deficiency

The objective of this study was to characterize levodopa (l‐dopa)–induced dyskinesias in patients with tyrosine hydroxylase deficiency. Clinical observation was carried out on 6 patients who were diagnosed with tyrosine hydroxylase deficiency and were treated with escalating doses of l‐dopa. All 6 patients showed l‐dopa‐induced dyskinesias of variable intensity early in the course of treatment and regardless of the age of initiation. l‐Dopa–induced dyskinesias were precipitated by increases in the dose of l‐dopa and also by febrile illnesses and stress. They caused dysfunction and distress in 2 patients. The dyskinesias were improved by decreasing the l‐dopa dose or by slowing its titration upward. Increasing the dose frequency was helpful in 2 patients, and introducing amantadine was helpful in another 2 patients. l‐Dopa–induced dyskinesias are a common phenomenon in tyrosine hydroxylase deficiency. The current observations show that l‐dopa–induced dyskinesias are frequent in a dopamine‐deficient state in the absence of nigrostriatal degeneration. Although l‐dopa–induced dyskinesias in tyrosine hydroxylase deficiency are phenomenologically similar to those that occur in Parkinsons disease, they are different in a number of other respects, suggesting intrinsic differences in the pathophysiologic basis of l‐dopa–induced dyskinesias in the 2 conditions.

Homovanillic acid in cerebrospinal fluid of 1388 children with neurological disorders.

To determine the prevalence of dopaminergic abnormalities in 1388 children with neurological disorders, and to analyse their clinical, neuroradiological, and electrophysiological characteristics.

Secondary abnormalities of neurotransmitters in infants with neurological disorders.

Neurotransmitters are essential in young children for differentiation and neuronal growth of the developing nervous system. We aimed to identify possible factors related to secondary neurotransmitter abnormalities in pediatric patients with neurological disorders. We analyzed cerebrospinal fluid (CSF) and biogenic amine metabolites in 56 infants (33 males, 23 females; mean age 5.8mo [SD 4.1mo] range 1d–1y) with neurological disorders whose aetiology was initially unknown. Patients were classified into three clinical phenotypes: epileptic encephalopathy, severe motor impairment, and non‐specific manifestations. All patients showed normal results for screening of inborn errors of metabolism. We report clinical, neuroimaging, and follow‐up data. Among the patients studied, 10 had low homovanillic acid (HVA) levels and in four patients, 5‐hydroxyindoleacetic acid (5‐HIAA) was also reduced. Patients with neonatal onset had significantly lower levels of HVA than a comparison group. HVA deficiency was also associated with severe motor impairment and the final diagnosis related to neurodegenerative disorders. 5‐HIAA values tended to be decreased in patients with brain cortical atrophy. The possibility of treating patients with L‐Dopa and 5‐hydroxytryptophan, in order to improve their neurological function and maturation, may be considered.

Methods for the diagnosis of creatine deficiency syndromes: A comparative study

The increasing number of patients with creatine deficiency syndromes (CDS) stresses the need to develop screening procedures for the identification these inherited disorders. Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of CDS and several analytical methods to measure both metabolites have been developed. High-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is quick and sensitive but, unlike HPLC and gas chromatography-mass spectrometry (GC/MS), it is unavailable in most laboratories. Thus, we decided to evaluate comparatively HPLC-MS/MS, GC/MS and HPLC methods, as well as to establish reference values in a healthy paediatric population. According to our results, these three methods may be suitable for analysing GAA in urine. Furthermore, Passing-Bablock plots showed good agreement among all three. However, when comparing the Cr/Crn ratio, our results revealed that while HPLC-MS/MS data were in agreement with those of GC/MS, a constant and proportional error was observed when compared with those of HPLC. Consequently, the Cr/Crn ratio obtained by the last method should be evaluated with caution. Our reference values for GAA and Cr/Crn ratio in urine negatively correlate with age. Concerning GAA and Cr measurements in plasma, it is interesting to note that in contrast to what was occurring in urine, GAA concentration increased significantly with age, while we did not find any significant difference for Cr values within the same age group.