Reduction of large neutral amino acid levels in plasma and brain of hyperleucinemic rats

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Abstract

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly known. In the present study we investigated the effect of acute hyperleucinemia on plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected subcutaneously with 6 μmol l-leucine per gram body weight. Controls received saline in the same volumes. The animals were sacrificed 30–120 min after injection, blood was collected and their brain rapidly removed and homogenized. The amino acid concentrations were determined by HPLC using orthophtaldialdehyde for derivatization and fluorescence for detection. The results showed significant reductions of the large neutral amino acids (LNAA) l-phenylalanine, l-tyrosine, l-isoleucine, l-valine and l-methionine, as well as l-alanine, l-serine and l-histidine in plasma and of l-phenylalanine, l-isoleucine, l-valine and l-methionine in brain, as compared to controls. In vitro experiments using brain slices to study the influence of leucine on amino acid transport and protein synthesis were also carried out. l-Leucine strongly inhibited [14C]-l-phenylalanine transport into brain, as well as the incorporation of the [14C]-amino acid mixture, [14C]-l-phenylalanine and [14C]-l-lysine into the brain proteins. Although additional studies are necessary to evaluate the importance of these effects for MSUD, considering previous findings of reduced levels of LNAA in plasma and CSF of MSUD patients during crises, it may be speculated that a decrease of essential amino acids in brain may lead to reduction of protein and neurotransmiter synthesis in this disorder.

Introduction

The branched chain amino acid and organic acid disorder maple syrup urine disease (MSUD), caused by a severe deficiency in activity of the branched chain ketoacid dehydrogenase complex (EC 1.2.4.4), is biochemically characterized by the tissue accumulation of the branched chain amino acids (BCAA) l-leucine (Leu), l-isoleucine (Iso) and l-valine (Val) and the corresponding branched chain keto acids (BCKA) l-alpha-ketoisocaproate (KIC), l-alpha-keto-beta-methylvalerate (KMV) and l-alpha-ketoisovalerate (KIV) (Chuang and Shih, 1995). Serum Leu and KIC concentrations usually reach 5 mmol/L during crises, concentrations much higher than those of the other accumulating metabolites (Langenbeck et al., 1978). Clinically, and according to its response to thiamine administration, MSUD can be classified into five groups (Chuang and Shih, 1995). Patients with the classical form show their first signs during the neonatal period, presenting severe neurological deterioration and convulsions, and generally die within months if treatment is not instituted. A marked increase of serum and urine concentrations of BCAA and BCKA is the biochemical hallmark of the disorder. The other variants are characterized by a milder phenotype, with lesser accumulation of the characteristic metabolites, but the affected patients usually present a variable degree of psychomotor delay or mental retardation.

The underlying etiopathogenic mechanisms leading to neurological dysfunction in MSUD are not well understood. However, increased plasma concentrations of Leu are associated with the appearance of neurologic symptoms and Leu and/or its ketoacid KIC have been considered the main neurotoxic metabolites in MSUD (Snyderman et al., 1964, Efron, 1965, Chuang and Shih, 1995). On the other hand, it has been postulated that brain energy deficit especially during crises may contribute to brain injury. KIV and KIC acid reduce respiration in rat brain slices (Howell and Lee, 1963, Danner and Elsas, 1989) and KIC inhibits pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activities in rat and human brain mitochondria at concentrations within the range of BCKA accumulation in the blood of MSUD patients during metabolic decompensation (Land et al., 1976, Danner and Elsas, 1989). Moreover, it has been reported that KIV, KIC and their hydroxyderivatives compete with l-glutamate for decarboxylation in rat brain homogenates reducing gamma-aminobutyric acid (GABA) concentration (Tashian, 1961). On the other hand, genetic animal models of MSUD in calves demonstrated that affected subjects present a marked loss in number of postsynaptic GABAA receptors, as assessed in [3H]diazepam binding studies (Harper et al., 1986, Dodd et al., 1992).

We have recently observed that MSUD patients present a reduction of the large neutral amino acids (LNAA) l-methionine (Met), l-tryptophan, l-phenylalanine (Phe) and l-tyrosine (Tyr) in plasma and CSF during crises of metabolic decompensation, when higher tissue levels of Leu and the other branched-chain amino acids were found. We are tempted to speculate that the blood decrease of these essential amino acids may result in the deficit of these essential amino acids in the brain as well as of their products such as proteins and neurotransmitters, a fact that might contribute to the neurological dysfunction of MSUD (Wajner et al., 2000, Wajner and Vargas, 1999).

In the present paper we report the effect of acute injection of Leu into young rats on the plasma and cerebral concentrations of amino acids, in the hope to understand the alterations of LNAA levels in plasma of MSUD patients and to test whether Leu injection alters the concentrations of these essential substances in the brain. In order to achieve plasma Leu levels similar to those encountered in human MSUD, we used a chemical model of acute hyperleucinemia recently reported by our laboratory (Mello et al., 1999).

Section snippets

Subjects and reagents

Fifteen-day-old Wistar rats of both sexes from our breeding stock were used. The animals were maintained on a 12-h light/dark cycle (lights on at 07:00 h) in a constant-temperature colony room (25±2°C) with water and standard lab chow (Supra, Porto Alegre) ad lib. Pregnant rats were housed in individual cages and left undisturbed throughout gestation. Twenty-four hours after delivery all pups were placed together and eight pups were assigned randomly to each dam. At 15 days of age, the animals

Results

Unless otherwise stated, the tables exhibit only the plasma and brain concentrations of the amino acids showing significant differences from the controls. The plasma amino acid levels at 30, 60, 90 and 120 min after Leu or cycloleucine injection are shown in Table 1. After Leu injection, there was a statistically significant increase of plasma Leu levels which was accompanied by a significant decrease in the plasma concentrations of Met, Val, Iso, Phe, Tyr, His, Ser and Ala, more evident at 60

Discussion

MSUD is an inherited metabolic disorder characterized by severe impairment of CNS function (Chuang and Shih, 1995). Although some neurochemical effects of the metabolites appearing in high concentrations in MSUD have been reported, the mechanisms of neurotoxicity of the disorder are not understood well (Tashian, 1961, Howell and Lee, 1963, Land et al., 1976). However, Leu and its alpha-keto acid KIC are the major metabolites accumulating in the disorder and, for this reason, are considered to

Acknowledgements

This work was supported by grants from PRONEX II, CNPq, FAPERGS and PROPESQ/UFRGS. We also thank Mr Fabrı́cio Gomes Barbosa for technical support.

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