Background: The significance of plasma l-alloisoleucine, which is derived from l-isoleucine in vivo, for diagnosis of maple syrup urine disease (MSUD) was examined. Methods: Branched-chain l-amino acids were measured by automatic amino acid analysis. Results: Alloisoleucine reference values in plasma were established in healthy adults [1.9 ± 0.6 μmol/L (mean ± SD); n = 35], children 3–11 years (1.6 ± 0.4 μmol/L; n = 17), and infants <3 years (1.3 ± 0.5 μmol/L; n = 37). The effect of dietary isoleucine was assessed in oral loading tests. In controls receiving 38 μmol (n = 6; low dose) and 1527 μmol (n = 3; high dose) of l-isoleucine per kilogram of body weight, peak increases of plasma isoleucine were 78 ± 24 and 1763 ± 133 μmol/L, respectively; the peak increase of alloisoleucine, however, was negligible for low-dose (<0.3 μmol/L) and minor for high-dose (5.5 ± 2.1 μmol/L) load. In patients with diabetes mellitus, ketotic hypoglycemia, phenylketonuria, and obligate heterozygous parents of MSUD patients, alloisoleucine was not significantly different from healthy subjects. Therefore, a plasma concentration of 5 μmol/L was used as a cutoff value. In patients with classical MSUD (n = 7), alloisoleucine was beyond the cutoff value in 2451 of 2453 unselected samples. In patients with variant MSUD (n = 9), alloisoleucine was >5 μmol/L in all samples taken for establishment of diagnosis and in 94% of the samples taken for treatment control (n = 624). With the other branched-chain amino acids, the frequency of diagnostically significant increases was <45%. Conclusions: The present findings indicate that plasma l-alloisoleucine above the cutoff value of 5 μmol/L is the most specific and most sensitive diagnostic marker for all forms of MSUD.

L-alloisoleucine (2S, 3R), a diastereomer of L-isoleucine (2S, 3S), is a normal constituent of human plasma. Considerable amounts accumulate in maple syrup urine disease, in which the branched-chain 2-oxo acid dehydrogenase step is impaired. The mechanism of L-alloisoleucine formation, however, is unclear. We addressed this issue by performing oral L-[1-13C]isoleucine loading (38 micromol/kg body wt, 50% 1-13C) in overnight-fasted healthy subjects (n = 4) and measuring the 3-h kinetics of 13C-label incorporation into L-isoleucine plasma metabolites. Compared with L-isoleucine, the time course of 13C-enrichment in the related 2-oxo acid, S-3-methyl-2-oxopentanoate, was only slightly delayed. Peak values, amounting to 18+/-4 and 17+/-3 mol percent excess, respectively, were reached within 35 and 45 min, respectively. The kinetics of 13C-enrichment in S- and R-3-methyl-2-oxopentanoate enantiomorphs were similar and linearly correlated (p << 0.001). In L-alloisoleucine, however, 13C-label accumulated only gradually and in minor amounts. Our results indicate that R-3-methyl-2-oxopentanoate is an immediate and inevitable byproduct of L-isoleucine transamination and further suggest that alloisoleucine is primarily formed via retransamination of 3-methyl-2-oxopenanoate in vivo.

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We characterized the effect of orthotopic liver transplantation on the catabolism of branched-chain L-amino acids in a female patient with classical form of maple syrup urine disease. Transplantation was performed at the age of 7.4 years due to a terminal liver failure triggered by a hepatitis A infection. Since then, the patient is on an unrestricted diet and plasma concentrations of branched-chain L-amino and 2-oxo acids are stable, yet at moderately increased levels (2- to 3-fold of control). L-Alloisoleucine concentrations, however, remained remarkably elevated (> 5-fold of control). In vivo catabolism was investigated by measuring the metabolic L-alloisoleucine clearance and whole-body leucine oxidation in the postabsorptive state. In an oral loading test with 580 micromol alloisoleucine per kg body wt, the L-alloisoleucine elimination rate constant (0.067 h(-1)) was in the normal range (0.069+/-0.012 h(-1), n = 4). In an oral L-[1-13C]leucine load (38 micromol/kg body wt), 19.5% of the tracer dose applied was recovered in exhaled 13CO2 versus 18.9+/-3.6% in healthy subjects (n = 10). Thus, the patient exhibited obviously normal whole-body catabolic rates although branched-chain L-amino acid oxidation was confined to the liver transplant. Most likely, the enhanced substrate supply from extrahepatic sources led to an elevation of the plasma concentrations and thus induced a compensatory enhancement of the metabolic flux through the branched-chain 2-oxo acid dehydrogenase complex in the intact liver tissue.