Does hyperphenylalaninemia induce brain glucose hypometabolism? Cerebral spinal fluid findings in treated adult phenylketonuric patients.

Despite numerous studies in human patients and animal models for phenylketonuria (PKU; OMIM#261600), the pathophysiology of PKU and the underlying causes of brain dysfunction and cognitive problems in PKU patients are not well understood. In this study, lumbar cerebral spinal fluid (CSF) was obtained immediately after blood sampling from early-treated adult PKU patients who had fasted overnight. Metabolite and amino acid concentrations in the CSF of PKU patients were compared with those of non-PKU controls. The CSF concentrations and CSF/plasma ratios for glucose and lactate were found to be below normal, similar to what has been reported for glucose transporter1 (GLUT1) deficiency patients who exhibit many of the same clinical symptoms as untreated PKU patients. CSF glucose and lactate levels were negatively correlated with CSF phenylalanine (Phe), while CSF glutamine and glutamate levels were positively correlated with CSF Phe levels. Plasma glucose levels were negatively correlated with plasma Phe concentrations in PKU subjects, which partly explains the reduced CSF glucose concentrations. Although brain glucose concentrations are unlikely to be low enough to impair brain glucose utilization, it is possible that the metabolism of Phe in the brain to produce phenyllactate, which can be transported across the blood-brain barrier to the blood, may consume glucose and/or lactate to generate the carbon backbone for glutamate. This glutamate is then converted to glutamine and carries the Phe-derived ammonia from the brain to the blood. While this mechanism remains to be tested, it may explain the correlations of CSF glutamine, glucose, and lactate concentrations with CSF Phe.

Retrospective analysis of 19 patients with 6-Pyruvoyl Tetrahydropterin Synthase Deficiency: Prolactin levels inversely correlate with growth.

BACKGROUND: Pyruvoyl Tetrahydropterin Synthase (PTPS) Deficiency is the most common form of BH4 deficiency resulting in hyperphenylalaninemia. It can have variable clinical severity and there is limited information on the clinical presentation, natural history and effectiveness of newborn screening for this condition. METHODS: Retrospective data (growth and clinical parameters, biochemical and genetic testing results, treatment) were collected from 19 patients with PTPS deficiency in different centers, to evaluate biochemical and clinical outcomes. Descriptive statistics was used for qualitative variables, while linear regression analysis was used to correlate quantitative variables. RESULTS: Patients with PTPS deficiency had an increased incidence of prematurity (4/18) with an average gestational age only mildly reduced (37.8 ± 2.4 weeks) and low birth weight (-1.14 ± 0.97 SD below that predicted for gestational age). With time, weight and height approached normal. VALUES: All patients were identified by newborn screening for an elevated phenylalanine level. However, phenylalanine levels were normal in two whose testing was performed at or before 24 h of age. Sapropterin dihydrochloride treatment normalized phenylalanine levels. Molecular testing identified novel variants in the PTS gene, some of which present in more than one affected family. The neurotransmitter derivatives 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in the CSF were decreased in most cases except in 2 families with the peripheral form of PTPS deficiency. With time, HVA and 5HIAA became abnormally low in two of these patients requiring therapy. Prolactin (whose secretion is inhibited by dopamine) levels were elevated in several patients with PTPS deficiency and inversely correlated with the z-scores for height (p < 0.01) and weight (p < 0.05). Most patients with PTPS deficiency had delayed development early in life, improving around school age with IQs mostly in the normal range, with a small decline in older individuals. From a neurological standpoint, most patients had normal brain MRI and minor EEG anomalies, although some had persistent neurological symptoms. DISCUSSION: Patients with PTPS deficiency have not only an increased incidence of prematurity, but also decreased birth weight when corrected for gestational age. Hyperphenylalaninemia can be absent in the first day of life. Therapy with sapropterin dihydrochloride normalizes phenylalanine levels and neurotransmitter precursors can improve CSF neurotransmitter metabolites levels. Insufficient dopaminergic stimulation (as seen from elevated prolactin) might result in decreased height in patients with PTPS deficiency. Despite early delays in development, many patients can achieve independence in adult life, with usually normal neuroimaging and EEG.

Chiral Capillary Electrophoresis-Mass Spectrometry.

Capillary electrophoresis (CE) is a well-established and one of the most powerful separation techniques in the field of chiral separations. Its hyphenation with mass spectrometry (MS) combines both the high separation efficiency and low sample consumption of CE and the high sensitivity and structural information of MS. Thus, the outstanding chiral resolution power of CE along with the MS advantages makes CE-MS a perfect combination to achieve sensitive enantioseparations. This chapter describes three representative examples of different approaches used in the chiral analysis of amino acids in biological fluids by CE-MS. The first methodology uses the partial filling technique to avoid the entry of cyclodextrins in the MS source. The second method shows the possibility to carry out the direct coupling EKC-MS even when a relative high concentration of a native cyclodextrin is used as chiral selector. The last example illustrates an alternative strategy based on the formation of stable diastereomers between an enantiomerically pure chiral reagent and the amino acids enantiomers which can be separated in an achiral environment.

Alterations in the tyrosine and phenylalanine pathways revealed by biochemical profiling in cerebrospinal fluid of Huntington's disease subjects.

Huntington's disease (HD) is a severe neurological disease leading to psychiatric symptoms, motor impairment and cognitive decline. The disease is caused by a CAG expansion in the huntingtin (HTT) gene, but how this translates into the clinical phenotype of HD remains elusive. Using liquid chromatography mass spectrometry, we analyzed the metabolome of cerebrospinal fluid (CSF) from premanifest and manifest HD subjects as well as control subjects. Inter-group differences revealed that the tyrosine metabolism, including tyrosine, thyroxine, L-DOPA and dopamine, was significantly altered in manifest compared with premanifest HD. These metabolites demonstrated moderate to strong associations to measures of disease severity and symptoms. Thyroxine and dopamine also correlated with the five year risk of onset in premanifest HD subjects. The phenylalanine and the purine metabolisms were also significantly altered, but associated less to disease severity. Decreased levels of lumichrome were commonly found in mutated HTT carriers and the levels correlated with the five year risk of disease onset in premanifest carriers. These biochemical findings demonstrates that the CSF metabolome can be used to characterize molecular pathogenesis occurring in HD, which may be essential for future development of novel HD therapies.

The effect of L-dopa infusions with and without phenylalanine challenges in parkinsonian patients: plasma and ventricular CSF L-dopa levels and clinical responses.

We monitored the motor response and plasma and ventricular CSF (CSFv) concentrations of L-dopa during IV infusions of L-dopa in two patients with advanced Parkinson's disease. Concentrations of L-dopa in CSFv mirrored, but lagged behind, those in plasma. In the fasting state, the duration, but not the magnitude, of the motor response was greater with increasing plasma and CSFv levels of L-dopa. During IV infusions of L-dopa following oral administration of phenylalanine, a large neutral amino acid that shares a transport system into the brain with L-dopa, the duration of the motor response was markedly attenuated despite undiminished CSFv levels of L-dopa. These observations suggest that either L-dopa entry into CSFv and the brain are differentially affected by phenylalanine or that phenylalanine affects other steps in the motor response. These observations demonstrate that, except in the fasting state, L-dopa in CSFv is not a reliable predictor of motor response.

Correlation between cerebrospinal fluid phenylalanine and beta-endorphin in patients with phenylketonuria.

Previous animal and human studies have suggested an analgesic effect of phenylalanine involving endogenous opioid peptides. Phenylalanine was measured by a HPLC method with electrochemical detection and beta-endorphin by a specific radioimmunoassay in 14 lumbar cerebrospinal fluid samples from 13 patients with phenylketonuria. Cerebrospinal fluid beta-endorphin was also determined in 6 age-matched control subjects. We found a trend towards a higher beta-endorphin level in phenylketonuria (median 26.0 pM, range 13.0-37.8) than in the control subjects (20.6 pM, 12.7-28.0), P = 0.13. Cerebrospinal fluid concentrations of phenylalanine and beta-endorphin were significantly correlated (r = 0.68, P = 0.008). The results support the hypothesis that phenylalanine modifies the central endogenous opioid system.

Neurometabolic effects of ACTH on free amino compounds in opsoclonus-myoclonus syndrome.

To evaluate the possible role of central free amino compounds in pediatric opsoclonus-myoclonus syndrome (OMS), 21 cerebrospinal fluid (CSF) amino compounds were measured by an amino acid analyzer or mass spectroscopy in 74 anesthetized children, 54 with OMS and 20 age-matched neurological controls. In OMS, only phosphoethanolamine was increased compared to controls; OMS severity and duration had significant converse effects on alanine and phosphoethanolamine. In contrast, corticotropin (ACTH) treatment was associated with increased alanine and phenylalanine, and decreased taurine compared to controls and untreated OMS, and increased glutamine, lysine, ornithine, and tyrosine compared to untreated OMS. Other than low taurine, these effects were not found with corticosteroid treatment, and non-steroidogenic immunotherapy had no effect. The ACTH dose-association was most apparent for alanine and phosphoethanolamine, but lysine and ornithine were also higher in the high-dose ACTH group. There were no significant disease- or treatment-associated perturbations in GABA, glycine, or other amino acids. These data suggest a unique pattern of ACTH effects on non-neurotransmitter CSF amino compounds, for the most part not shared by steroids.

Content of phenylalanine, tyrosine and their metabolites in CSF in phenylketonuria.

By using ion-exchange chromatography and gas chromatography coupled with mass spectrometry, the content of phenylalanine, tyrosine and their metabolites typical of phenylketonuria (PKU) was determined in the cerebrospinal fluid (CSF) of 8 untreated children with classical PKU and 9 controls. At the same time, plasma and urine were analysed. In PKU the content of phenylalanine is increased on average 23 times in plasma and CSF. The content of phenylalanine and tyrosine in CSF is about 4 times less as compared with plasma. The phenylalanine-to-tyrosine ratio is approximately the same for these fluids both in control and in PKU. This indicates that the transport of phenylalanine and tyrosine through the blood-brain barrier is not disturbed in PKU. Phenylpyruvate and 4-hydroxyphenylpyruvate are either not detected or present in very low concentrations in the CSF of children with PKU; their derivatives, phenyllactate and 4-hydroxyphenyllactate, are present in relatively higher concentrations. This indicates increased metabolic conversion in brain tissues.

Increased concentrations of tyrosine and phenylalanine in the cerebrospinal fluid as a possible reflection of related changes of the brain tissue in newborns with intra-uterine growth retardation.

Plasma and cerebrospinal fluid concentrations of tyrosine and phenylalanine were measured fluorometrically in human term neonates appropriate for gestational age and in neonates small for gestational age with severe intra-uterine growth retardation of Type II. The intra-uterine retarded newborns showed significant differences of both plasma and cerebrospinal fluid tyrosine levels as compared with the non-retarded newborns: the mean plasma and cerebrospinal fluid tyrosine were determined to be 2.0 and 2.5 times, respectively, higher in the retarded group. The mean cerebrospinal fluid phenylalanine content was also increased in these newborns. The findings may reflect cellular disturbances of the tyrosine metabolization in the developing brain as it can also be found in experimental intra-uterine growth retarded animals.

On the brain barrier system function and changes of cerebrospinal fluid concentrations of phenylalanine and tyrosine in human phenylketonuria.

In 6 patients with classic phenylketonuria (PKU) the plasma and cerebrospinal fluid (CSF) concentrations of phenylalanine and tyrosine were measured fluorimetrically. The results of the PKU group were compared with data obtained from 17 children without abnormal CSF parameters and free of metabolic or central nervous disorders, in whom a diagnostic lumbal puncture has been performed. The PKU patients showed statistically significant differences in comparison with the controls: plasma and CSF phenylalanine contents were markedly higher (on the average 6.4 and 4.6 times, respectively) in PKU patients. Plasma tyrosine was 1.8 times lower, but CSF tyrosine was about 2.2 times higher in comparison to the controls. In general, the plasma CSF-ratio ( PLR ) of phenylalanine did not change in PKU and could be found in the same range as in the normal controls. In contrast to this, the PLR of tyrosine was found to be significantly lower in PKU patients. The results are discussed with respect to an altered function of the brain barrier systems for the amino acid transport in PKU, and that increased CSF tyrosine contents in PKU may rather reflect disturbances of the intracellular metabolism of the brain cells than changes of the amino acid transport through the brain barrier produced by hyperphenylalaninemia.

Elevated levels of amino acids in the CSF of motor neuron disease patients.

Amino acid levels in the cerebrospinal fluid of patients with motor neuron disease were compared with an age-matched control group receiving diagnostic myelography. Five amino acids were significantly elevated in the cerebrospinal fluid of the motor neuron disease patients compared to the controls. These were isoleucine, glycine, alanine, phenylalanine, and threonine, which were increased by 60, 58, 38, 26, and 25% respectively. A significant increase was also obtained when the amino acids with nonpolar R groups were grouped together (34%). The significance of these findings is discussed in terms of amino acid changes that occur in both normal aging and other neurological conditions.

Reduction of cerebrospinal fluid phenylalanine after oral administration of valine, isoleucine, and leucine.

A supplement of the branched chain amino acids, valine, isoleucine, and leucine (VIL) was administered orally to patients with phenylketonuria, either together with unrestricted diet of natural protein or with a low phenylalanine diet. The VIL supplement brought about a significant reduction of the cerebrospinal fluid-serum ratio of phenylalanine from a mean value of 0.254 without VIL to 0.204 with VIL. The reduction varied from 15-40% (mean 21%). Concentrations of glycine, lysine, methionine, threonine, tryptophan, and tyrosine were within normal limits in serum and cerebrospinal fluid of infants with phenylketonuria. No amino acid imbalance was created by the supplement and no adverse effects from VIL were observed.

Precursors and metabolites of phenylethylamine, m and p-tyramine and tryptamine in human lumbar and cisternal cerebrospinal fluid.

Phenylacetic acid, p-hydroxyphenylacetic acid, m-hydroxyphenylacetic acid, phenylalanine, indoleacetic acid, 5-hydroxyindoleacetic acid and tryptophan were measured in lumbar and cisternal cerebrospinal fluid (CSF) taken during pneumoencephalography. The data suggest that the concentration of the acid metabolites of the trace amines tryptamine, phenylethylamine, p-tyramine and m-tyramine in lumbar CSF are influenced by the system that transports these acids out of CSF. In cisternal CSF this mechanism does not operate and more information can be obtained on the metabolism of the parent amines in the CNS. Our data indicate that (1) m-tyramine is relatively unimportant quantitatively (2) the rate of metabolism of phenylethylamine in human brain is similar to that of 5-hydroxytryptamine (3) the most important variable controlling the synthesis of phenylethylamine is the activity of aromatic amino acid decarboxylase (4) p-tyramine is synthesised at about half the rate of phenylethylamine and is thus quantitatively important in metabolic terms.

Plasma and cerebrospinal fluid amino acid concentrations in phenylketonuria during the newborn period.

Plasma and cerebrospinal fluid amino acid values were determined in 29 infants 9 to 30 days of age with a confirmed diagnosis of phenylketonuria. Phenylalanine concentrations in plasma and cerebrospinal fluid were markedly elevated; the degree of elevation in the cerebrospinal fluid had a significant relationship to that of the plasma. The only other significant deviations in the plasma were reductions in the threonine and tyrosine values. Cerebrospinal fluid threonine, alanine, and arginine concentrations were reduced, whereas those of serine, isoleucine, and histidine were elevated. This combined deficiency and excess of amino acids in the central nervous system may have a significant effect on protein synthesis at a time in life when this synthesis and turnover is most active.

The fate of N-dansyl-L-phenylalanine in the cerebrospinal fluid after intraventricular and intracisternal injection: a comparative fluorescence microscopic and analytical study.

Absorption, accumulation and release of N-Dansyl-L-phenylalanine (DPA) through the ependyma, plexus choriodei and brain parenchyma after intraventricular and intracisternal injection was examined at different postinjection intervals by fluorescence microscopy. The following results were obtained: 1. After intraventricular injection, DPA is rapidly absorbed from the ependyma and plexus choriodei in all ventricles and subsequently disappears from the various points of the ventricles at different times. DPA is no longer evident in the ependyma after 40 min and the plexus after 90 min. Aborption and storage occur primarily in the dopaminergic centers of the brain. This stage begins 5 min p.i. attains a maximum after 40 min and is maintained up to 180 min p.i. 2. If DPA is administered intracisternally, fluorescence is initially restricted to the ependyma and choroid plexus of the fourth ventricle and to the wall of the aquaeduct. Only at 5-10 min p.i. are rostral ventricular portions labelled. Passage of the amino acid out of the ventricle only occurs to a limited extent. 40 min after intracisternal injection, DPA is no longer demonstrable in the ependyma and plexus or brain parenchyma. 3. Intrathecally administered DPA appears in the periglomerular tubules of the kidney as well 2.5 min p.i. and is stored there for up to 40 min. The kidney medulla remains free of fluorescence. 4. DPA injected into the CSF is protein-bound.

A new theory of enterorecirculation of amino acids and its use for depleting unwanted amino acids using oral enzyme-artificial cells, as in removing phenylalanine in phenylketonuria.

Oral binders remove intestinal bile acid and prevent its reabsorption and recycling thereby lowering systemic cholesterol levels. The results in this paper demonstrate the presence of another extensive enterorecirculation for amino acids. Pancreatic and other glandular secretions into the intestine contain large amounts of proteins, enzymes and polypeptides. Tryptic digestion converts these into amino acids which are then reabsorbed back into the body as they pass down the intestine. This paper shows that this forms a large enterorecirculation of amino acids between the body and intestine. The dietary protein source of amino acids is negligible when compared to the endogenous source, since this paper shows that protein-free diet did not alter the intestinal amino acid concentration. This raises the possibility of using this for the selective depletion of specific body amino acids. In this paper we use a phenylketonuria (PKU) model in rats to test the use of this hypothesis. In PKU rats, artificial cells microencapsulated phenylalanine ammonia lyase (PAL) given orally is more effective than a phenylalanine-free diet. The enzyme artificial cells are more efficient in lowering PHE in the intestine, plasma and cerebrospinal fluid. Compared to PKU on PHE-free diet, this has resulted in better weight gain and general physical condition. Preliminary studies also show that artificial cells microencapsulated asparaginase, glutaminase and tyrosinase given orally can deplete the corresponding amino acid from the intestine.