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J. Inherit. Metab. Dis. 23 (2000) 505È512 ( SSIEM and Kluwer Academic Publishers. Printed in the Netherlands

Reduction of large neutral amino acid concentrations in plasma and CSF of patients with maple syrup urine disease during crises M. WAJNER1,2*, D. M. COELHO1, A. G. BARSCHAK1, P. R. ARAU JO1,2, R. F. PIRES1,2, F. L. G. LULHIER1 and C. R. VARGAS1,2 1 ServicÓ o de Gene tica Me dica, Hospital de Cl• nicas de Porto Alegre, Porto Alegre RS ; 2 Departamento de Bioqu• mica, Instituto de Cieü ncias Ba sicas de Sau de, UFRGS, Porto Alegre, RS, Brazil * Correspondence : Departamento de Bioqu• mica, Instituto de Cieü ncias Ba sicas da Sau de, UFRGS, Rua Ramiro Barcelos 2600, CEP 90.035-003, Porto Alegre, RS, Brazil. E-mail MW AJNER=V ORT EX.UFRGS.BR MS received 6.07.99

Accepted 7.01.00

Summary : Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly understood. We determined the concentrations of all amino acids in plasma of patients with MSUD during crises (with severe CNS symptoms) and after recovery in the hope of detecting possible alterations of these levels during metabolic decompensation. Blood samples obtained from 11 children with MSUD aged 1 month to 7 years and from 10 age-matched controls (5 months to 6 years) with no evidence of metabolic disease were examined for their amino acid content by high-performance liquid chromatography. We observed that leucine, isoleucine and valine concentrations were respectively 30, 9 and 3 times higher than normal values, whereas the concentrations of the large neutral amino acids (LNAA) phenylalanine, tyrosine, tryptophan and methionine were signiÐcantly lower during metabolic decompensation as compared to the controls. In addition, concentrations of leucine, but not of valine or isoleucine, were inversely related to the LNAA concentrations in plasma. The concentrations of these amino acids in plasma returned to normal values when patients were clinically well. CSF amino acid concentrations also showed decreased amounts of LNAA and increased concentrations of branched-chain amino acids. It is possible that the decrease in plasma concentrations of LNAA may lead to a deÐcit of these essential amino acids in the brain as well as of their products such as proteins and neurotransmitters, a fact that might be related to the neurological dysfunction of MSUD. 505

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Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by a severe deÐciency in the activity of the branched-chain ketoacid dehydrogenase complex (BCKAD ; EC 1.2.4.4) (Chuang and Shih 1995). It is biochemically characterized by the accumulation of the branched-chain amino acids (BCAAs) leucine (Leu), isoleucine (Ile) and valine (Val) and the corresponding branched-chain keto acids a-ketoisocaproate (KIC), a-keto-b-methylvalerate (KMV) and aketoisovalerate (KIV). Most patients with the classical form present with severe neurological deterioration and convulsions, and generally die within months when treatment is not instituted. Although the pathophysiology of the neurological dysfunction of MSUD is poorly understood, there is a large body of evidence associating defective leucine metabolism and the neurological symptoms of these patients (Efron 1965 ; Snyderman et al 1964). Increased plasma concentrations of leucine are associated with the appearance of neurological symptoms and leucine and/or its ketoacid, aketoisocaproate, have been considered the main neurotoxic metabolites in MSUD (Chuang and Shih 1995). We report here the concentrations of free amino acids in plasma of patients a†ected by classical MSUD during metabolic decompensation and after recovery with dietary therapy and compare these concentrations with those of 10 agematched controls. A signiÐcant reduction of plasma concentrations of various large neutral amino acids (LNAA) was observed during decompensation. After recovery, the amino acid concentrations returned to normal levels. We discuss these Ðndings and attempt to relate them to the characteristic neurological dysfunction of the disorder. Some of these results have appeared in letter form (Wajner and Vargas, 1999). MATERIALS AND METHODS Materials : We studied 11 children with MSUD (nine with the classical and two with the intermittent form), aged 1 month to 7 years, admitted to hospital during decompensation episodes with convulsions, hypotonia, coma, ketonaemia/ketonuria and metabolic acidosis. Crises occurred following infections in nine children, whereas in two infants they corresponded to the Ðrst decompensation episode. Venous blood specimens (1È5 ml) were collected on di†erent occasions from all patients during crises and from Ðve of these children following their discharge from hospital. CSF was also taken from one infant with classical MSUD with no diagnosis at the time of collection. All patients were in good nutritional state, but a variable degree of psychomotor delay/mental retardation was seen in six. Plasma from 10 aged-matched children (aged 5 months to 6 years) who were fasted to exclude metabolic disease was analysed as controls. None showed evidence of metabolic disease. Samples were prepared for analysis immediately after collection. Methods : The free amino acids in plasma and CSF were determined as follows. The samples were deproteinized by adding 200 kl methanol to 50 kl plasma or CSF and the sediment was removed by centrifugation (400g for 15 min). The analysis was performed by reversed-phase high-performance liquid chromatography (HPLC) and J. Inherit. Metab. Dis. 23 (2000)

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Ñuorescent detection using orthophthaldialdehyde plus mercaptoethanol. The Ñow rate was 1.5 ml/min in a gradient of the mobile phase (methanol and phosphate bu†er : bu†er A, 80% methanol ; bu†er B, 20% methanol), and the duration of each analysis was 45 min (Joseph and Mardsden 1986). The amino acids were quantitatively determined by relating their chromatographic peak areas to those obtained from a known standard mixture and to that of the internal standard homocysteic acid. Serum insulin concentrations were measured by radioimmunoassay (RIA, kit, CIS) and serum glucose by the glucose oxidase method (Glucose Oxidase kit, Diagnostica Merck). Data were analysed statistically by the MannÈWhitney U-test and calculations were performed on a PC-compatible computer using the Statistical Package for the Social Sciences (SPSS) system. RESULTS Table 1 shows the results obtained by quantitative amino acid analysis of plasma specimens obtained from 11 MSUD patients during crises and from 10 children of similar age (control group). For the calculations we used the plasma specimen showing the highest leucine concentration during decompensation from each Table 1 Plasma concentrations of amino acids (lmol/L) in control and MSUD patients during crises Controls

MSUD patients

Amino acid

Mean

SEM

Range

Mean

SEM

Range

Asp Glu Ser His Gln Gly ] Thr Ala Tyr Trp Met Val Phe Ile Leu Orn Lys

35.1 84.6 134.7 46.0 368.0 206.7 319.5 126.9 39.0 43.7 268.9 77.6 84.2 121.0 64.4 243.7

10.9 22.3 18.0 11.7 40.2 9.1 57.6 11.0 6.6 11.0 36.0 18.2 14.6 15.3 17.6 54.1

2.6È120.5 9.9È213.9 49.1È225.6 5.0È94.6 233.7È658.0 163.7È452.8 36.0È562.4 77.6È184.7 4.8È83.8 14.9È112.4 120.5È460.8 14.4È185.9 21.9È155.1 64.8È198.3 7.7È200.5 84.0È546.3

9.68 363.3 155.4 25.6 443.5 241.4 243.6 65.8* 4.47** 7.6* 757.1** 7.65** 699.8** 3271.4** 40.3 159.4

3.7 140.2 16.9 9.3 129.3 27.7 72.5 19.7 3.1 3.2 85.2 4.6 115.4 506.6 15.3 79.8

NDÈ30.0 11.4È1266.2 64.7È213.4 NDÈ75.3 68.3È1271.6 209.1È403.2 24.7È666.6 8.7È229.1 NDÈ31.4 NDÈ25.1 300.9È1110.0 NDÈ34.5 262.7È1234.1 1015.9È4318.8 NDÈ154.4 NDÈ808.6

Values represent mean ^ SEM. Number of MSUD patients \ 11. Number of controls \ 10. Comparison between means of controls and MSUD patients calculated by the MannÈWhitney U-test ; * p \ 0.05 ; ** p \ 0.01 Asp, aspartate ; Glu, glutamate ; Ser, serine ; His, histidine ; Gln, glutamine ; Gly, glycine ; Thr, threonine ; Ala, alanine ; Tyr, tyrosine ; Trp, tryptophan ; Met, methionine ; Val, valine ; Phe, phenylalanine ; Ile, isoleucine ; Leu, leucine ; Orn, ornithine ; Lys, lysine. ND, not detected.

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patient. The normal ranges reported in the literature are the same as those indicated for the controls of the same age. The table shows that there was an especially marked increase in plasma concentrations of leucine, isoleucine and valine in MSUD patients, which were respectively 30, 9 and 3 times higher than controls. In contrast, plasma concentrations of the large neutral amino acids tyrosine, tryptophan, phenylalanine and methionine were signiÐcantly diminished. A nonsigniÐcant decrease in plasma aspartate and an increase in plasma glutamic acid concentrations was also found in MSUD patients. Since samples from some patients were obtained on various occasions, it was possible to perform a longitudinal study. Table 2 compares the plasma amino acid values obtained for MSUD patients during crises of metabolic decompensation and after recovery. For the calculations we used the plasma specimen showing the highest leucine during decompensation and the lowest value after recovery. It can be seen that the concentrations of branched-chain amino acids were signiÐcantly reduced to nearly normal when patients were clinically well, and this was accompanied by normalization of the concentrations of the other amino acids whose concentrations were diminished during crises. Figure 1 shows the individual plasma values of leucine, tryptophan, tyrosine, methionine and phenylalanine in one patient during an episode of decompensation and with treatment. An inverse relationship can be observed between the plasma concentrations of leucine and of the other LNAA studied. The coefficients of correlation between leucine and the other amino acids were r \ [0.8432, p \ 0.01 (tryptophan), r \ [0.8197, p \ 0.01 (tyrosine), r \ [0.7144, p \ 0.01 (phenylalanine) and r \ [0.8832, p \ 0.05 (methionine). In Table 2 Plasma concentrations of amino acids (lmol/L) in MSUD patients during metabolic decompensation and after recovery During crises

After crises

Amino acids

Mean

Range

Mean

Range

Glu Ser His Gln Gly ] Thr Ala Tyr Trp Met Val Phe Ile Leu Orn Lys

484.8 163.1 19.7 499.7 275.0 258.1 91.1 8.0** 8.3* 801.2** ND 853.3** 3654.5** 56.4 191.5

21.3È1266.2 64.7È213.4 NDÈ53.3 153.9È936.3 209.1È403.2 45.6È666.6 29.9È229.1 NDÈ31.4 NDÈ25.1 300.9È1110.0 ND 262.7È1234.1 1956.3È6022.1 NDÈ154.4 NDÈ808.6

119.8 172.8 27.9 526.1 395.2 248.1 120.0 33.4 15.1 151.3 38.2 160.5 711.3 51.3 176.7

14.9È238.7 33.3È321.3 NDÈ61.7 92.5È1497.1 130.8È628.5 54.3È540.5 24.4È311.9 4.6È138.2 4.9È30.6 58.3È347.7 NDÈ63.6 15.3È346.8 187.7È2638.9 6.2È85.5 75.9È251.1

Values represent means and ranges. Number of MSUD patients \ 5. Comparison between means of groups calculated by the MannÈWhitney U-test ; * p \ 0.05 ; **p \ 0.01 Abbreviations as in Table 1

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Figure 1 Plasma leucine and large neutral amino acid concentrations in one MSUD patient during treatment. The values correspond to plasma amino acid levels from one MSUD patient during metabolic decompensation and after recovery. Leu, leucine ; Phe, phenylalanine ; Tyr, tyrosine ; Met, methionine ; Trp, tryptophan

addition, leucine correlated positively with isoleucine (r \ 0.6905, p \ 0.05), but not with valine (r \ 0.4069, p [ 0.05), and valine and isoleucine did not correlate with methionine and the aromatic amino acids. LNAA concentrations in CSF were also diminished, as revealed in one patient during decompensation, whereas the concentrations of the branched-chain amino acids were greatly elevated (Table 3). Insulin and glucose concentrations were also measured in serum from MSUD patients during crises and from age-matched controls. Insulin levels (kIU/mL) were 4.1 ^ 2.1 (n \ 5) (MSUD) and 12.8 ^ 3.3 (n \ 10) (controls) (p \ 0.05) respectively, whereas glucose levels (mg/100 ml) were 68.5 ^ 32.2 (n \ 5) and 82.3 ^ 12.3 (n \ 10) (p \ 0.20), respectively. Table 3 Cerebrospinal Ñuid concentrations of LNAA (lmol/L) in a MSUD infant during crisis Amino acid

MSUD patient

Normal values (1È12 months)

Tyr Trp Met Val Phe Ile Leu

ND ND ND 130.3 1.5 29.6 263.0

4.1È14.0 0È2.2 1.0È4.2 9.1È20.1 3.4È15.0 3.1È5.7 7.3È14.5

Abbreviations as in Table 1

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DISCUSSION The underlying pathogenic mechanisms leading to neurological dysfunction in MSUD are not well understood. However, it is believed that leucine and its aketoacid, a-ketoisocaproate (KIC), which accumulate most in the disorder, are the principal neurotoxic agents (Chuang and Shih 1995 ; Efron 1965 ; Snyderman et al 1964). In addition, it has been postulated that brain energy deÐcit, especially during crises, may contribute to brain injury. a-Ketoisovaleric acid (KIV) and KIC acid reduce respiration rate in rat brain slices (Danner and Elsas 1989 ; Howell and Lee 1963) ; KIC inhibits pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activities in rat and human brain mitochondria at BCKA concentrations within the range present in the blood of MSUD patients during metabolic decompensation (Danner and Elsas 1989 ; Land et al 1976). 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 (Danner and Elsas 1989 ; Tashian 1961). BCAA and their keto acids have also been shown to interfere with myelin development and especially the synthesis of proteolipid protein by inhibiting protein synthesis in brain slices (Appel 1966). In the present study we observed that, besides the characteristically elevated plasma concentrations of the BCAA leucine, isoleucine and valine, most of the large neutral amino acids, namely tryptophan, tyrosine, methionine and phenylalanine, showed signiÐcantly reduced concentrations in plasma from patients during crises as compared to controls of the same age. These amino acids returned to nearly normal values after the patientsÏ recovery, when leucine and the other BCAA concentrations had signiÐcantly decreased. Our Ðndings agree with a study demonstrating a signiÐcant reduction of Tyr, Phe and Met in plasma of normal volunteers after intravenous infusion of leucine alone or of all the BCAA (Erikson et al 1981). The explanation for the decreased plasma concentrations of various LNAA could not be the poor intake of essential amino acids during decompensation leading to a decrease of these amino acids in plasma, since the age-matched controls were fasted and thus should also have shown reduced LNAA concentrations. Other possibilities could be the speciÐc stimulation of muscle protein synthesis by leucine and insulin (Buse and Reid 1975 ; Tovar et al 1991), or the activation of glutamate dehydrogenase activity in liver and skeletal muscle by leucine, which could result in increased amino acid utilization by these tissues (Eguchi et al 1992 ; Zhou and Thomson 1996), but this would be expected to result in an inÑux of all (essential and nonessential) amino acids into the peripheral tissues and a fall in their plasma concentrations. Furthermore, the present results show a signiÐcant diminution of insulin concentrations in blood of MSUD patients compared to the controls, in agreement with data reported by Haymond and colleagues (1973). Finally, another possibility could be the sequestration and/or accelerated catabolism of the amino acids appearing at low concentration in plasma of MSUD patients by peripheral tissues through LNAA competition with leucine for the efflux from these tissues through the L membrane carrier system, as previously shown in hyperphenylalaninaemia (Cespedes et al 1989 ; Christensen 1986, 1990 ; Oxender and Christensen 1963). It is interesting to J. Inherit. Metab. Dis. 23 (2000)

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note that we found a signiÐcant inverse correlation between plasma concentrations of leucine and of methionine, tyrosine, tryptophan and phenylalanine. Therefore, if the trapping e†ect of the BCAA, especially leucine, is a major mechanism for the decrease in plasma LNAA concentration in MSUD, it is reasonable to propose that these amino acids would leave the cells after the decrease of tissue leucine accumulation. On the other hand, considering that the brain concentrations of essential amino acids are determined largely by plasma concentrations and transport across the bloodÈbrain barrier (BBB) and that Phe and Leu use more than 50% of the transport sites of the L system in cerebrovascular cells at normal plasma levels (Fernstrom and Faller 1978 ; Lerner and Larimore 1986 ; Tews et al 1987), it is presumed that patients with PKU and MSUD, by having selective increases of plasma Phe and BCAA, respectively, have brain depletion of important essential LNAA needed for protein and neurotransmitter synthesis. This assumption is supported by recent Ðndings showing that Phe strongly inhibits the transport of other amino acids across the human BBB by the L carrier system (Shulkin et al 1995), by the reduced brain uptake of methionine-C11 in PKU (Comar et al 1981), by the depletion of amino acids in the brain found at autopsy in PKU patients (McKean et al 1968), by the diminution of the concentrations of various LNAA in brain and plasma of rats receiving a diet rich in leucine, with higher depletion occurring in brain Phe, Tyr and Met (Block and Harper 1990), and by our present Ðndings of diminished concentrations of the LNAA in the CSF of a MSUD patient. In conclusion, we presume that the reduced plasma concentrations of several essential amino acids in MSUD patients reported here may further aggravate the synthesis of important brain components and contribute to the neurological dysfunction characteristic of these patients. In this context, we should emphasize the importance of supplementing MSUD patients with the other LNAA that compete with Leu, Ile and Val and that are diminished in their blood, especially during crises. ACKNOWLEDGEMENT This study was supported by FIPE/HCPA, CCNPq, FINEP, FAPERGS and PROPESP/UFRGS. We are very grateful for the technical assistance of Mr Fabrizio G. Barbosa. REFERENCES Appel SH (1966) Inhibition of brain protein synthesis : an approach to a biochemical basis of neurological dysfunction in the amino-acidurias. T rans NY Acad Sci 29 : 63È70. Block KP, Harper AE (1990) High levels of dietary amino and branched-chain a keto acids alter plasma and brain amino acid concentrations in rats. J Nutr 121 : 663È671. Buse MG, Reid SS (1975) LeucineÈa possible regulator of protein turnover in muscle. J Clin Invest 56 : 1250È1261. Cespedes C, Thoene JG, Lowler K, Christensen HN (1989) Evidence for inhibition of exodus of small neutral amino acids from non-brain tissues in hyperphenylalaninaemia rats. J Inherit Metab Dis 12 : 166È180. Christensen HN (1986) Where do the depleted plasma amino acids go in phenylketonuria ? Biochem J 233 : 929È930.

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