Renal excretion of galactose and galactitol in patients with classical galactosaemia, obligate heterozygous parents and healthy subjects.

The age dependence of galactose and galactitol excretion was assessed in overnight-fasted galactose-1-phosphate uridyltransferase-deficient patients under dietary treatment (ages 4-34 years; n = 51), obligate heterozygous parents (ages 25-71 years; n = 49) and healthy subjects (ages 3-58 years; n = 215). Urine concentrations were analysed by stable-isotope dilution gas chromatography mass spectrometry. There was considerable interindividual variability. The intraindividual variation, however, was not age-dependent and was rather low. Excretion estimates were calculated from the creatinine-related concentrations using weight-, age- and sex-related creatinine excretion rates. Experimental evidence is presented underscoring the problems inherent in random sampling and substantiating the primary endogenous origin of galactose and galactitol in postabsorptive urine samples. Age-dependent excretion estimates were best fitted to a simple growth-related model assuming an exponential decrease with age until adulthood. According to the model, mean postabsorptive galactose and galactitol excretion in healthy subjects was similar and decreased exponentially from about 1.2 micromol/kg body weight per day in infants to about 0.2 micromol/kg body weight per day in adults. Excretion in heterozygotes was normal. In galactosaemic patients, galactose excretion was in the normal range. Galactitol excretion, however, was enhanced over 50-fold and decreased from a mean estimate of about 64 micromol/kg body weight per day in infants to about 23 micromol/kg body weight per day in adults. The results are discussed with respect to the significance of galactose and galactitol excretion for whole-body galactose removal and with respect to the applicability of urinary galactitol analysis for metabolic monitoring in galactosaemia.

Whole-body L-leucine oxidation in patients with variant form of maple syrup urine disease.

Whole-body L-leucine oxidation was assessed in patients with maple syrup urine disease of different severity using oral L-[1-(13)C]leucine bolus tests (38 micromol/kg body weight). Residual whole-body L-leucine oxidation was estimated on the basis of the 3-h kinetics of (13)CO(2) exhalation and (13)C-isotopic enrichment in plasma 4-methyl-2-oxopentanoate using a noncompartmental mathematical approach. In four patients with classical maple syrup urine disease (two females and two males; mean age, 13 +/- 5 y; range, 7--17 y), L-leucine oxidation was too low to be measurable. In two females (aged 11 and 15 y) with a severe variant form of the disease, whole-body L-leucine oxidation was reduced to about 4% of control. In six milder variants (two females and four males; mean age +/- SD, 15 +/- 10 y; range, 6--34 y), the estimates for residual whole-body L-leucine oxidation ranged from 19 to 86% (59 +/- 24%) of control and were substantially higher than the residual branched-chain 2-oxo acid dehydrogenase complex activities in the patients' fibroblasts (10--25% of control). Possible mechanisms are considered that might contribute to a comparatively high residual in vivo L-leucine oxidation in (mild) variant maple syrup urine disease.

Analysis of concentration and (13)C enrichment of D-galactose in human plasma.

BACKGROUND: A stable-isotope dilution method for the sensitive determination of D-galactose in human plasma was established. METHODS: D-[(13)C]Galactose was added to plasma, and the concentration was measured after D-glucose was removed from the plasma by treatment with D-glucose oxidase and the sample was purified by ion-exchange chromatography. For gas chromatographic-mass spectrometric analysis, aldononitrile pentaacetate derivatives were prepared. Monitoring of the [MH-60](+) ion intensities at m/z 328, 329, and 334 in the positive chemical ionization mode allowed the assessment of 1-(12)C-, 1-(13)C-, and U-(13)C(6)-labeled D-galactose, respectively. The D-galactose concentration was quantified on the basis of the (13)C-labeled internal standard. RESULTS: The method was linear (range examined, 0.1-5 micromol/L) and of good repeatability in the low and high concentration ranges (within- and between-run CVs <15%). The limit of quantification for plasma D-galactose was <0.02 micromol/L. Measurements in plasma of postabsorptive subjects yielded D-galactose concentrations (mean +/- SD) of 0.12 +/- 0.03 (n = 16), 0.11 +/- 0.04 (n = 15), 1.44 +/- 0.54 (n = 10), and 0.17 +/- 0.07 (n = 5) micromol/L in healthy adults, diabetic patients, patients with classical galactosemia, and obligate heterozygous parents thereof, respectively. These data were considerably lower (3- to 18-fold) than the values of a conventional enzymatic assay. The procedure was also applied successfully in a stable-isotope turnover study to evaluate endogenous D-galactose formation. CONCLUSIONS: The present findings establish that detection of D-galactose from endogenous sources is feasible in human plasma and show that erroneously high results may be obtained by enzymatic methods.

Formation of L-alloisoleucine in vivo: an L-[13C]isoleucine study in man.

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.

Significance of L-alloisoleucine in plasma for diagnosis of maple syrup urine disease.

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 micromol/L (mean +/- SD); n = 35], children 3-11 years (1.6 +/- 0.4 micromol/L; n = 17), and infants <3 years (1.3 +/- 0.5 micromol/L; n = 37). The effect of dietary isoleucine was assessed in oral loading tests. In controls receiving 38 micromol (n = 6; low dose) and 1527 micromol (n = 3; high dose) of L-isoleucine per kilogram of body weight, peak increases of plasma isoleucine were 78 +/- 24 and 1763 +/- 133 micromol/L, respectively; the peak increase of alloisoleucine, however, was negligible for low-dose (<0.3 micromol/L) and minor for high-dose (5. 5 +/- 2.1 micromol/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 micromol/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 micromol/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 micromol/L is the most specific and most sensitive diagnostic marker for all forms of MSUD.

Renal clearance of branched-chain L-amino and 2-oxo acids in maple syrup urine disease.

In maple syrup urine disease (MSUD), branched-chain L-amino (BCAA) and 2-oxo acids (BCOA) accumulate in body fluids owing to an inherited deficiency of branched-chain 2-oxo acid dehydrogenase complex activity. In MSUD, little information is available on the significance of urinary disposal of branched-chain compounds. We examined the renal clearance of leucine, valine, isoleucine and alloisoleucine, and their corresponding 2-oxo acids 4-methyl-2-oxopentanoate (KIC), 3-methyl-2-oxobutanoate (KIV), (S)-(S-KMV), and (R)-3-methyl-2-oxopentanoate (R-KMV), using pairs of plasma and urine samples (n = 63) from 10 patients with classical MSUD. The fractional renal excretion of free BCAA was in the normal range (< 0.5%) and independent of the plasma concentrations. The excretion of bound (N-acylated) BCAA was normal and not significantly dependent on the BCAA plasma concentrations. The fractional renal excretion of BCOA was in the order KIC << KIV < R-KMV < or = S-KMV (range (%): KIC 0.1-25; KIV 0.14-21.3; S-KMV 0.26-24.6; R-KMV 0.1-35.9), significantly correlated with the KIC plasma concentrations, and generally higher than that of the related BCAA. The results show that the renal excretion of free BCAA as well as of the acylated derivatives is negligible. The renal excretion of BCOA, however, to some extent counteracts increases in BCAA concentrations and thus contributes to the lowering of total BCAA pools in MSUD.

Assessment of whole body L-leucine oxidation by noninvasive L-[1-13C]leucine breath tests: a reappraisal in patients with maple syrup urine disease, obligate heterozygotes, and healthy subjects.

Suitability of a recently proposed noninvasive L-[13C]leucine breath test for assessment of whole body leucine oxidation in maple syrup urine disease (MSUD) was examined. Oral L-[1-13C]leucine loads (38 micromol/kg body weight) were performed in overnight fasted MSUD patients (n = 6, classical form), obligate heterozygote parents (n = 6), and control subjects (n = 10). Three-hour 13CO2 exhalation kinetics were evaluated using curve fitting procedures. Venous blood was obtained in most cases and analyzed for 13C-labeled plasma metabolites. In control subjects, maximal 13CO2 exhalation was reached at tmax = 55 +/- 18 min. Cumulative 13CO2 output at 3 h amounted to 4.7 +/- 0.7 micromol x (kg body weight)(-1). Estimated total 3CO2 exhalation was 7.2 +/- 1.4 micromol x (kg body weight)(-1) (19.0 +/- 3.6% of the dose). Half of this amount was expired at t1/2 = 130 +/- 18 min. The data show a considerable degree of intersubject variability. Intraindividual variability was comparable, however, when checked in two volunteers. In obligate heterozygotes, 13CO2 kinetics were similar to controls (tmax = 35 +/- 8 min, t1/2 = 95 +/- 16 min). Total 13CO2 output [5.7 +/- 1.4 micromol x (kg body weight)(-1)] tended to be in the lower control range. None of the MSUD patients under study exhibited a significant increase in 13CO2 output after load. Maximal increase of label in plasma 4-methyl-2-oxopentanoate, the physiologic precursor of 13CO2, was 16.1 +/- 3.5 MPE in control subjects. In MSUD, label dilution was increased and correlated with the patients' leucine/4-methyl-2-oxopentanoate plasma levels. Considering the generally high variability of 13CO2 output and the unstable substrate pools in MSUD, we discuss the limitations of whole body leucine oxidation measurements by noninvasive approaches.

Determination of R- and S-3-methyl-2-oxopentanoate enantiomers in human plasma: suitable method for label enrichment analysis.

A sensitive method for the determination of S- and R-3-methyl-2-oxopentanoate enantiomers (KMV, alpha-keto-beta-methylvalerate) in physiological fluids suitable for isotope enrichment analysis is described: after extraction with acid, 2-oxo acids are separated from interfering amino acids by cation-exchange chromatography. Reductive amination of the branched-chain 2-oxo acids by use of L-leucine dehydrogenase yields the corresponding L-amino acids. L-Isoleucine and L-alloisoleucine which are formed from S- and R-3-methyl-2-oxopentanoate, respectively, are then quantified by amino acid analysis. The method was used for determination of the R-IS-3-methyl-2-oxopentanoate ratio in plasma of healthy subjects and patients with diabetes mellitus and maple syrup urine disease. Applicability for gas chromatographic-mass spectrometric analysis of 13C-label enrichment in plasma S-3-methyl-2-oxopentanoate is demonstrated.

Enzymatic-chemical preparation of quinoxaline derivatives from L-amino acids for gas chromatographic-mass spectrometric analyses.

We report on an enzymatic-chemical method for the specific preparation of L-amino acid-derived quinoxalinols suitable for the sensitive estimation of 13C- or 2H-label enrichment by gas chromatography-mass spectrometry: Amino acid fractions are isolated from physiological fluids by ion-exchange chromatography. The compound of interest is converted to the corresponding 2-oxo acid by treatment with an L-amino acid dehydrogenase of the desired specificity. Reaction of the 2-oxo acid with o-phenylenediamine yields the quinoxaline derivative. Isotopic label enrichment is then determined by gas chromatographic-mass spectrometric analysis of the O-trimethylsilyl derivative using an ammonia-chemical ionization mode and selected ion monitoring of the quasi-molecular ions [MH]+ and [MH + n]+ (n = number of labeled positions). Details for application of various generally available L-amino acid dehydrogenases (L-ala DH, L-glu DH, L-leu DH, L-phe DH) are presented. The method was used, e.g., for serum analysis in in vivo studies on the decarboxylation rates of branched-chain L-[1-(13)C]-amino acids.

Human branched-chain L-amino acid aminotransferase: Activity and subcellular localization in cultured skin fibroblasts.

Assay conditions for measurement of human skin fibroblast branched-chain L-amino acid aminotransferase activity were established and applied to studies on subcellular distribution and kinetic properties of the enzyme. Digitonin fractionation of cultured cells revealed that the aminotransferase activity was mainly (at least about 95%) associated with mitochondrial citrate synthase activity. As tested with L-leucine, activity of the enzyme against amino group acceptors (forward reaction) was in the order 2-oxoglutarate [Symbol: see text] branched-chain > straight-chain 2-oxo acids (C3-C8). With 4-methyl-2-oxopentanoate, activity against amino group donors (reverse reaction) was in the order L-glutamate [Symbol: see text] branched-chain > straight-chain (C2-C6) and other L-amino acids. The data suggest that, in human fibroblasts, isoenzyme type I resides within the mitochondrial space. Possible implications for the metabolism of branched-chain compounds are discussed.

On the mechanism of L-alloisoleucine formation: studies on a healthy subject and in fibroblasts from normals and patients with maple syrup urine disease.

L-Alloisoleucine formation from L-isoleucine was studied in vitro and in vivo. When a healthy subject was loaded with L-isoleucine, plasma levels of L-isoleucine and 3-methyl-2-oxopentanoate (KMV), as well as L-alloisoleucine, increased. Peak values were reached successively and were in the order L-isoleucine much greater than KMV much greater than L-alloisoleucine. Metabolic clearance of L-isoleucine and KMV was rapid; clearance of L-alloisoleucine was considerably delayed. When human skin fibroblast cultures were challenged with L-isoleucine, KMV accumulated at a gradually decreased rate, whereas L-alloisoleucine accumulated at a gradually accelerated rate. KMV and L-alloisoleucine formation were related and depended on the L-isoleucine concentration applied. In cell lines derived from MSUD patients (classical form), metabolite formation was only about 2-fold higher than in control strains. The relatively small difference between normal and MSUD fibroblasts in vitro as opposed to the striking differences between healthy subjects and MSUD patients in vivo are discussed with respect to the significance of physiological mechanisms participating in the formation and degradation of L-alloisoleucine in man.

Transamination and oxidative decarboxylation of L-isoleucine, L-alloisoleucine and related 2-oxo acids in perfused rat hind limb muscle.

Metabolism of L-isoleucine, L-alloisoleucine and corresponding 2-oxo acids in rat hind limb muscle was comparatively studied under steady-state perfusion conditions. At 0.5 mM L-[1-14C]isoleucine, apparent transamination and 2-oxo acid decarboxylation rates amounted to about 17 and 4 nmol/min per g of muscle, respectively. With L-allo[1-14C]isoleucine, the corresponding rates were about 5- and 10-fold lower, respectively. After addition of dichloroacetate (1-5 mM), the portion of (S)- and (R)-methyl-2-oxopentanoate undergoing further oxidative decarboxylation within the tissue was similarly increased by over 40%. In perfusions with 0.5 mM (R,S)-3-methyl-2-oxopentanoate and tracer doses of 1-14C-labeled (S)- or (R)-enantiomer, the 14CO2 production was comparable (about 0.5 nmol/min per g of muscle). Dichloroacetate caused a several-fold increase in 14CO2 release from either enantiomer, apparent 2-oxo acid transamination rates remaining unaffected. Indications for a racemization of 2-oxo acid were not obtained in the experiments. The results are discussed with respect to the appearance/disappearance of L-alloisoleucine in vivo and to the fact that (R)-3-methyl-2-oxopentanoate, but not L-alloisoleucine, can support growth of rats on a diet deficient in L-isoleucine.

Transamination and oxidative decarboxylation rates of branched-chain 2-oxo acids in cultured human skin fibroblasts.

Transamination and oxidative decarboxylation of branched-chain L-amino acid derived 2-oxo acids in cultured human skin fibroblasts from normal subjects and from a patient with maple syrup urine disease (variant form) were comparatively studied in incubations with 1-14C-labeled substrates (1 mmol/liter). With normal cells, 14CO2 release ranged from about 11 to 3 nmol/90 min/mg of cell protein in the order 3-methyl-2-oxo[14C]butanoate greater than (S)-3-methyl-2-oxo[14C]pentanoate greater than 4-methyl-2-oxo[14C]pentanoate greater than (R)-3-methyl-2-oxo[14C]pentanoate. Formation of the corresponding branched-chain amino[14C] acids was substantially higher than 14CO2 production (around 10-fold) and similar with L-valine, L-isoleucine, and L-leucine. L-Allo-isoleucine production [from (R)-3-methyl-2-oxopentanoate] was significantly lower. With maple syrup urine disease fibroblasts, comparable transamination rates were observed. Related to the findings with normal cells, 14CO2 release from each substrate was differently reduced and apparent residual branched-chain 2-oxo acid dehydrogenase complex activity with 3-methyl-2-oxobutanoate, 4-methyl-2-oxopentanoate, (S)-, and (R)-3-methyl-2-oxopentanoate amounted to 12, 13, 22, and 50%, respectively.