Cerebral biochemical abnormalities in experimental maternal phenylketonuria: gangliosides and sialoglycoproteins.

The present study sought a biochemical explanation for retarded brain development in the heterozygous offspring of the phenylketonuric (PKU) mother. Two rat models of simulated maternal PKU, one induced by p-chlorophenylalanine and phenylalanine and the other by phenylacetate, were employed in this investigation. Maternal PKU had no influence on cerebral concentrations of DNA, protein, and cholesterol, which were normal in the 2 d old pup. However, there was a noticeable disruption of the normal ganglioside pattern and a significant reduction of sialoglycoproteins. Concomitant with a delayed drop in the gangliosides Q1b and D3, was a slower rise in M1 and D1a. At least 66% of sialoglycoproteins located on SDS-PAGE gel chromatograms, by radioactivity incorporated in vivo from radiolabeled N-acetylmannosamine and by (3H) sialic acid released by Neuraminidase from periodate-(3H)borohydride labeled glycoproteins, have mobilities of the cell adhesion molecules N-CAM and D-CAM. Whether the reduction of the sialoglycoproteins induced by maternal PKU is mainly in these cell adhesion molecules requires further investigation. Interference with the function of gangliosides and certain sialoglycoproteins during cerebral development may contribute to the brain dysfunction observed in the offspring of PKU mothers not on diet control during pregnancy.

A biochemical explanation of phenyl acetate neurotoxicity in experimental phenylketonuria.

The in vivo formation of [1-14C]acetyl-coenzyme A from D-[3-14C]3-hydroxybutyrate in the brain of the suckling rat was not affected by postnatal exposure to phenyl acetate. However, utilization of the generated acetyl-coenzyme A was significantly inhibited in certain metabolic reactions, namely synthesis of fatty acids and of sterols, but not in others as the Krebs cycle reactions that lead to the production of dicarboxylic amino acids. The incorporation of D-[U-14C]glucosamine into N-acetylneuraminic acid bound to glycoproteins was appreciably diminished in the rat pup previously exposed to maternal phenylketonuria induced by phenyl acetate. During the period of very rapid development of the brain, interference by phenyl acetate and/or its metabolites with certain critical biosynthetic pathways that require acetyl-coenzyme A would significantly contribute to retarded maturation of the brain that occurs in phenylketonuria.

On the possible mechanism of phenylacetate neurotoxicity: inhibition of choline acetyltransferase by phenylacetyl-CoA.

The influence of phenylacetate, phenylbutyrate, and phenylacetyl-CoA on the activity of choline acetyltransferase and S-acetyl-CoA synthetase was investigated in vitro. Phenylacetyl-CoA was found to be a very potent inhibitor of choline acetyltransferase, competitive for acetyl-CoA with Ki of 3.1 X 10(-7)M. In contrast, millimolar concentrations of phenylacetate and phenylbutyrate were required to inhibit the activity of the enzyme. Activity of S-acetyl-CoA synthetase was affected only slightly by the three agents in concentrations of 10(-3)-10(-2)M. At this time, results are interpreted to suggest that in phenylketonuria, phenylacetate exerts its neurotoxic action through its metabolic product, phenylacetyl-CoA, which could severely decrease the availability of acetyl-CoA.

Biochemical indices of neuronal development in experimental phenylketonuria: high affinity transport systems and gangliosides.

High affinity transport systems and gangliosides were assessed in an animal model of experimental phenylketonuria, namely the rat injected with phenylacetate during the first 21 days of life. The velocity of synaptosomal high affinity uptake of [3H]-choline, [14C]-gamma-aminobutyric acid (GABA), and [14C]-glutamic acid served as a measure of the relative density of uptake sites of these specific types of terminals. A reduction of cholinergic (25-37%) and GABAergic (23-45%) functioning terminals was produced by phenylacetate in the hippocampal, occipital, and frontal cortices from 40- to 55- and 80- to 95-day-old rats. In contrast, glutamatergic terminals in these same areas of the cerebrum from animals of both ages were not affected. This selection effect of phenylacetate on synaptic junctions is discussed. Cerebral ganglioside content was reduced approximately 40% in experimental hyperphenylalaninemia induced with p-chlorophenylalanine and L-phenylalanine. A similar decrease was observed in the rat exposed to phenylacetate but not in the animal injected with phenylpyruvate. Short-term exposure to phenylacetate did not alter the capacity of the very young rat to utilize glucosamine for the biosynthesis and incorporation of sialic acid into gangliosides. The large decrease in cerebral ganglioside concentration and the significantly smaller percentage distribution of GM1, observed in the 19-day-old chronically exposed to phenylacetate, are apparently associated with deficient neuronal development.

Experimental maternal phenylketonuria: an examination of two animal models.

Two satisfactory rat models of maternal phenylketonuria (PKU) have been developed. Continuous subcutaneous infusion into pregnant rats from the 9th-20th day of gestation of either (1) phenylacetate (PA), to elevate plasma levels of unconjugated PA to 0.25-0.60 mumol/ml, or (2) a nontoxic dose (0.2 mumol/g/day) of p-chlorophenylalanine (pClPhe) with L-phenylalanine (Phe), to elevate plasma Phe levels at least 10-fold (1.7-2.3 mumol/ml) and unconjugated PA to at least 0.2 mumol/ml, produced the syndrome of untreated maternal PKU: spontaneous abortion, mortality rate greater than normal among the newborn, retarded growth of fetal body and brain, and a learning deficit among the progeny. From the plasma of rats infused with only pClPhe, a metabolite was isolated and identified as p-chlorophenylacetic acid. This compound, at a concentration greater than 0.15 mumol/ml plasma was found to retard fetal body and brain growth. In the pregnant rat, plasma levels of unconjugated PA, in the range observed in some PKU individuals on a normal diet, effectively induced a simulation of maternal PKU. The results of this investigation support our contention that PA, which is produced in excessive amounts in clinical PKU, is the primary cause of the brain dysfunction.

Experimental phenylketonuria: effect of phenylacetate intoxication on number of synapses in the cerebellar cortex of the rat.

In experimental phenylketonuria, induced in the rat by exposure to phenylacetate during the first 21 days of life, there was a significant reduction of boutons, a decrease of an average of 25% in the whole cerebellar molecular layer. Both the density of synaptic profiles per square unit and the number of synapses per unit volume were decreased in the phenylacetate-treated rat as compared to the age-matched control. Neuronal density was unaffected. Results are interpreted to show a deficit of synapses per neuron, probably due to a decrease in synaptic formation in phenylacetate-induced phenylketonuria. Undernutrition was eliminated as a contributing factor.

Purkinje cell dendritic development in experimental phenylketonuria. A quantitative analysis.

A comparison was made of cerebellar dendritic development in the normal rat and in a new model of phenylketonuria, the phenylacetate-treated suckling rat. Golgi stain analysis of the Purkinje cells shows striking regional variations in the dendritic growth. These variations were observed in both the control and phenylacetate-treated animals and were especially striking before 15 days of life. Quantitative analysis of the dendritic tree revealed, in the phenylacetate-treated rat, a significant reduction in the total number of dendritic branches. However, the individual terminal dendritic length was largely unaltered. These effects of phenylacetate differ from those of deafferentation, and starvation. Results of this investigation clearly define the harmful effects of phenylacetate on developing neurons and are compatible with the clinical observation that brain damage in phenylketonuria occurs mainly during the first few years of life, the critical period of neuronal development.

Neuropathology of phenylacetate poisoning in rats: an experimental model of phenylketonuria.

Results of this investigation indicate that the suckling rat treated with phenylacetate should be a useful new model for studying the pathogenesis of phenylketonuria and neuronal development. Both cerebellar and retinal neurons of postnatally treated rats are vulnerable to the adverse effects of phenylacetate. Morphological changes observed in the cerebellum, retina, and optic nerve of treated animals during the fourth to twenty-first days of life consist of regional reduction in the size of cerebellar vermis lobules IV, V, VIa, and IX, 35 to 40% reduction in thickness of the molecular layer, accumulation of cerebellar external granular cells and retinal neuroblastic cells, fewer parallel fibers in the cerebellar cortex, and fewer myelinated axons in the optic nerve.

Myelin deficiency in experimental phenylketonuria: contribution of the aromatic acid metabolites of phenylalanine.

Retarded body and brain growth and a deficit of myelin in the cerebral hemispheres and the cerebellum were observed in an animal model of phenylketonuria, the p-chlorophenylalanine and L-phenylalanine treated preweanling rat. These manifestations of phenylketonuria were reproduced in rats treated with phenylacetate in amounts approximating those likely to be produced in phenylketonuria. Young rats treated with equivalent amounts of other metabolites of phenylalanine, namely, phenylpyruvate, phenyllactate, and mandelate, which also accumulate in the brain during hyperphenylalaninemia, did not exhibit any toxic effects. Phenylpyruvate did not give rise to phenylacetate in the brain, but a small percentage was converted to phenyllactate. The gross composition of myelin isolated from the brains of saline and phenylacetate treated animals was similar. At various time intervals after subcutaneous injection, phenylacetate in the brain reached levels thirty times those of phenylpyruvate and phenyllactate, although animals received equivalent amounts of the three metabolites. The retarded growth of the body and brain of the young animal treated with phenylacetate may be attributed to the formation of phenylacetylcoenzyme A in the tissues. The site of action is very likely linked to acylcoenzyme A metabolism, i.e., the synthesis and utilization of acetylCoA and acetoacetylCoA, which are involved in reactions generating ATP and energy and in the synthesis of cholesterol and fatty acids. Results of this investigation indicate that growth retardation induced by phenylacetate during the period of very rapid development of the brain is responsible for the mental retardation in phenylketonuria.