Search

Phenylketonuria (PKU), the most frequent inborn error of metabolism (1/15,000 live births), is an autosomal recessive condition caused by phenylalanine hydroxylase deficiency. Despite early and strict dietary control, some PKU children still exhibit behavioral and cognitive difficulties suggestive of a partly prenatal brain injury. The reported variability between the cognitive and clinical phenotypes within the same family raises the question of modifying genes in PKU. We suggest here that monoamine oxidase type B, MAOB, an enzyme degrading phenylethylamine, a very toxic metabolite of phenylalanine, could act as a modifying gene since a variant enzymatic activity of MAOB in PKU patients with similar phenylalanine levels would result in different phenylethylamine levels and different clinical outcomes. Finally the report of low MAOB, and consequently expectedly high phenylethylamine levels in neonates is consistent with a phenylethylamine-mediated brain injury possibly causing irreversible damages in PKU newborns prior to onset of the low protein diet.

T lymphocytes express membrane antigen receptor TcR/CD3 complexes only when all subunits are correctly assembled. Studies on TcR/CD3 membrane negative T cell variants containing all necessary subunits intracellularly, may allow to identify amino acids important for different subunit interactions. In this review, we summarize our recent work on TcR/CD3 negative variants of the human T cell line Jurkat. We found two critical amino acids in the TcR-alpha and TcR-beta extracellular constant regions (phenylalanine n. 216 and intrachain disulfide cysteine n. 212) involved in TcR-alpha beta/CD3- gamma epsilon, delta epsilon intermediary complex/zeta 2 homodimer interactions: (1) amino acid exchanges of phenylalanine demonstrated the importance of an aromatic amino acid residue at this position; (2) the intrachain disulfide bond assures a tertiary structure of the constant domain that is necessary for association with zeta 2 homodimers.

Phenylketonuria is due in the very great majority of cases to a deficiency in phenylalanine hydroxylase, an enzyme whose cofactor is biopterin. Prenatal screening consists in measuring the concentration of phenylalanine in a sample of dried blood taken after birth (levels are already raised by day 3). Screening, organized by the Association française pour le dépistage et la prévention des handicaps de l'enfant, is very thorough (cover greater than 99%). Treatment involves observance of dietary restriction for at least five years. Results are good. Questions concerning the useful duration of dietary treatment, the level of phenylalanine that should not be exceeded, and the future of girls with PKU remain controversial. When adult, such girls may give birth to retarded children if they do not resume dietary restriction before becoming pregnant. Now that problems of screening, its organization, and the management of diet have been solved, these questions are the new challenge that faces us.

Hyperphenylalaninemias result from different enzymatic impairment, the most common and best studied which is phenylalanine hydroxylase (PAH) deficiency. The PAH gene has been cloned, sequenced and mapped: it is a single copy. Twenty-one mutations have now been characterized, but they constitute less than half of the haploid genotypes in French patients. A study of RFLP haplotypes is informative in 90% of families, but no linkage disequilibrium exists between illness and one particular haplotype. Prediction of phenotype from genotype seems possible, and could constitute a better therapeutic approach, perhaps including gene therapy in the most serious cases. The recently produced murine model should permit further progress to be made. Some hypotheses could be put forward about the origin and high frequency of this disease, that principally affects Caucasians: there is a consensus of opinion--though there is no definitive proof--that some selective advantage exists in individuals heterozygous for a PKU allele.

Cell cultures obtained from human uveal tumours have been used as experimental model to study the lethal effect consecutive to the neutron capture reaction on boron incorporated into cells as borophenylalanine. An irradiation with a neutron fluence of 6 x 10(9) n cm-2 reduced the number of viable cells by about 30%.

A reduction in the level of a new enzymatic assay--a phenyl valerate hydrolase (PVH)--has been found during the clinical evolution of toxic neuropathies (as almitrine-bismetilate ones) as well as alcoholic or diabetic neuropathies. The substrate and the enzymatic function are different from those used by M.K. Johnson for NTE. The method follows procedures comparable to NTE (differential determination after inhibition by paraoxon and by paraoxon plus mipafox or DFP). It may be useful to test possible neurotoxicity of drugs and chemicals.

The occurrence of increased levels of blood phenylalanine after therapeutic administration of folate analogues has been occasionally reported and attributed to the inhibition of dihydropteridine reductase, an enzyme maintaining the cofactor of phenylalanine hydroxylase in its active tetrahydrogenated form (tetrahydrobiopterin). To study further this metabolic effect, 46 patients receiving high dose methotrexate (5 to 8 g/m2) infusions have been studied. Significant increase in serum phenylalanine was observed in 95% of methotrexate cycles, occurring at the end of infusion. In contrast to the large inter-individual variations, maximal phenylalanine concentrations were of the same magnitude in each individual, suggesting individual predispositions. The hypothesis of an inhibition of dihydropteridine reductase by methotrexate was supported by the parallel course of serum biopterin and phenylalanine levels, but in some way contradicted by the rapid return to baseline values of both, 24 hours after the end of methotrexate infusion. This transient and often moderate hyperphenylalaniemia is probably harmless except if it reflects a more general inhibition of pteridine-dependent hydroxylases. Especially, such an inhibition of cerebral tyrosine- and tryptophan-hydroxylase activities might be the reason for transient neurological disturbances observed in some patients on high-dose methotrexate treatment.

Formation of toluene in growing cultures of Clostridium aerofoetidum strain WS was enhanced when the medium was supplemented with phenylacetic acid or with L-phenylalanine and L-methionine together. Evidence for the role of L-phenylalanine was shown by the detection of [2H2]-methyl[2,3,4,5,6-2H5]benzene ("heptadeuterotoluene") in growing cultures with L-[2',3',4',5',6'-2H5]phenyl[2,3-2H3]alanine and L-methionine.

One or two weeks after total mechanical occlusion of the dog colon, the organ is severely dilated, but the intraluminal pressure never exceeds 8 cm of water, and there are no obvious histological lesions of the mucosa. Perfusion of the occluded colon in vivo reveals marked inhibition of ion and water absorption; however, except in the case of potassium, net secretion as occurs in the occluded ileum does not develop. Glucose absorption in vivo and active phenylalanine and beta-methyl-D-glucoside transport in vitro are reduced in the mucosa of the occluded colon.

Descriptions of cases of PKU presenting the plain metabolic defect but little or no brain damage are becoming more and more frequent. The author admits the existence of a genetic enzyme system independent of the PKU gene (X). It is probably acting in the brain cells, restoring more or less normal aminoacid balance at this level. Full enzymatic activity and optimal protection against the deleterious effect of phenylalanine would result from the homogenous gene (XX), somewhat lower activity from the heterozygous gene (Xx). Homozygous xx-individuals would lack the protecting enzyme and therefore be highly exposed to damage by phenylalanine. Hydroxylation of phenylalanine in brain tissue has been reported for several animal species by a series of authors who incriminate either a brain isoenzyme of phenylalanine hydroxylase or tyrosine hydroxylase. The main aim of this paper is to suggest search for such types of enzyme in human brain cells to all the workers having access to human brain tissue.

Transaminase B (EC.2.6.1.6.) from E. coli, the specific enzyme for branched-chain aminoacids, was obtained in a purity equal to or greater than 96 p. cent after an 800-fold purification, employing two different procedures. One of the procedures involved heating at 60degreesC. The apparent molecular weight of the enzyme was estimated by chromatography on Sephadex and gel electrophoresis to be close to 180,000. The protein is made up of 6 subunits of equal size, with one molecule of coenzyme in each. Its absorption spectrum shows bands at 335 and 415 nm, and was found to be almost insensitive to the pH of the medium between 4.6 and 9. Transaminase B is active on phenylalanine as well, although the reaction between L-phenylalanine and alpha-ketoglutarate is about 50 to 100 times slower than the analogous reaction using L-valine as an aminoacid. Three sets of data show that the phenylalanine aminotransferase activity associated with transaminase B is not an artefact due to a protein contaminant. 1) Activities displayed toward phenylalanine and valine cannot be resolved by different methods, including chromatography, gel electrophoresis, and electrofucussing. 2) The absorption spectrum of the enzyme is as strongly modified by phenylalanine as by valine. 3) A ketoglutarate-free reaction between phenylalanine, tyrosine or typtophane and an aliphatic alpha-ketoacid is catalyzed by the pure enzyme and follows a mechanism belonging to the usual ping pong type. The possible significance of this reaction as a regulatory device in the cell metabolism is briefly discussed.

Chloramphenicol and erythromycin are antibiotics whose target is the bacterial ribosome. We have studied the possibility of isolating the bacterial ribosome by affinity chromatography on chloramphenicol and erythromycin columns. Several columns have been prepared using different spacers and methods of attachment of the ligands (cyanogen bromide, bis-expoxyde). The efficiency and specificity of these columns is discussed. Ethylene diamine is not always suitable as a spacer, because it presents non specific affinity for the ribosome. Pure tight ribosomes have been prepared by ultracentrifugation. They have been compared to ribosomes from affinity columns. These columns have no denaturing effect on ribosomes. They allow a good purification of ribosomes starting from crude bacterial extracts, but the separation of tight couples from loose subunits is not possible.

At least four separate enzymes are found to catalyze the transamination between phenylalanine and alpha-ketoglutarate in E. coli K 12, one of them being the aspartate aminotransferase. The Km of the latter enzyme for alpha-ketoglutarate is 0.3 or 0.035 mM according to the acceptor aminoacid being phenylalanine or aspartate respectively. The double specificity of aspartate aminotransferase in E. coli is however clearly shown by thermal inactivation studies using various effectors or different temperatures, and by the finding of an active transamination between aspartate and phenylpyruvate in the absence of ketoglutarate. This reaction shows the usual ping-pong type of mechanism, which implies that both substances are substrates for the same protein. Contrary to the phenylalanine-alpha-ketoglutarate reaction, which is probably of little importance in vivo when catalyzed by this enzyme, the direct ketoglutarate-free transamination between aspartate and the aromatic alpha-ketoacid is likely to represent a physiological function in regulating, at least partially, the balance between biosynthetic pathways for aromatic aminoacids and aspartate, for instance by maintaining similar ratios between the aminoacid and its ketoacid partner in both cases. For the sake of clarity it is proposed that the name "transaminase A", first devised by Rudman and Meister, be used for aspartate aminotransferase only, knowing that the specificity of this peculiar enzyme behaves as an accessory agent in the transamination of the aromatic compounds.

The specificity of phenylalanine and tyrosine carriers was investigated using actively metabolizing cells of Brevibacterium linens. The cellular protein synthesis of resting cells was very weakly inhibited, even with high concentrations of chloramphenicol or tetracycline. The nonaromatic amino acids were weak inhibitors for these carriers, while fluorinate analogues of phenylalanine and tyrosine were very potent competitive inhibitors. In practice these analogues cannot be used to replace amino acids to evaluate transport without incorporation because they are incorporated in cellular proteins.

A study of serum amino-acids during a L-phenylalanine loading test was performed in controls, heterozygotes and phenylketonuric patients with and without restricted diet. Abnormalities in plasma-amino acids were found in phenylketonuric patients at time zero with a fall of the level of some amino-acids (except for phenylalanine) when compared with the levels found in controls. These results are statistically valid only for threonine and proline. These abnormalities of the plasma amino-acids disappeared when patients were treated by a restricted diet. At the end of a loading test, there was no statistically valid changes of the blood amino-acids in controls, heterozygotes nor in patients with a restricted diet. There was no enhancement of the abnormalities in phenylketonuria.

We have presented the case of a 7 year old retarded child with psychotic like behavioral troubles. Biological studies are compatible with typical phenylketonuria. Two times, a low phenylalanine diet was followed by an improvement of the behavioral troubles, and the interruption of the diet by a relapse of these troubles. Biologically, an hyposerotoninergy was recorded by the lowering of the 5 HIAA renewal in CSF after probenecid, partially corrected by the low phenylalanine diet. From this personal case and a review of the literature, the authors discuss the biochemical interpretation of the behavioral troubles in the light of the serotoninergic deficit.

Since its description by Garrod, the concept of the "inborn errors of metabolism" has become a clinical reality that the physician cannot ignore. The aminoacidopathies represent a group historically notable in that they represent the first diseases recognized as metabolic in origin (alcaptonuria, albinism) and for which some measure of therapeutic success can be claimed. Dietary prophylaxis of the encephalopathy of phenylketonuria is a prominent example. Much progress has been made in the understanding of the aminoacidopathies that touch upon aspects as diverse as semiological study and prevention by pre- and neo-natal diagnosis. This brief overview presents the basic clinical, biochemical, genetic, physiopathological and therapeutic principles of these diseases. It provides representative examples of the diagnostic approach and draws particular attention to the need and the role of cross-disciplinary teamwork in the diagnosis and continuing care of the patient. While we wait for new methods of genetic correction, the main goal must be to prevent the clinical consequences of the metabolic defect.

An activator as well as an inhibitor of protein synthesis, were isolated from rabbit reticulocyte ribosomes. The ribosomes of the erythrocytes are inactive in protein synthesis and lacked the activator. However, when the activator moiety was added to the erythrocyte ribosomes, there was a significant increase in their capacity for protein synthesis, provided these ribosomes were freed of their inhibitor by washing beforehand with a detergent.