Hans-Werner Hammen

Longitudinal assessment of intellectual achievement in patients with classical galactosemia.

OBJECTIVE: To conduct a longitudinal assessment of long-term cognitive outcome in patients with classical galactosemia. METHODS: Inclusion criteria were (1) previous assessment of IQ dating back >10 years with tests being comparable with the recent German tests HAWIK-III and HAWIE-R, (2) absence of illnesses other than galactosemia, (3) absence of foreign language problems, (4) enzymatic-metabolic proof of classical galactosemia, (5) compliance with dietary therapy, and (6) written informed consent. Twenty-three patients who fulfilled these criteria were found. They underwent the first IQ test at a mean age of 11 ± 5 years and the second 13.6 to 15.5 years later at a mean age of 26 ± 5 years. Results were corrected for the obsolescence of test norms (Flynn effect). RESULTS: Mean total IQ scores on the first and second tests were 78 ± 14 and 73 ± 15, respectively, and not significantly different. IQ scores in the average range were observed for 7 patients on the first test and for 5 patients on the second test. For 17 patients, the intraindividual IQ scores remained essentially unchanged. Five patients showed a decrease and 1 an increase of the IQ score over time. No consistent pattern of change was found with respect to performance or verbal IQ subscores or in achievements in the individual subtest. CONCLUSIONS: The results confirm the presence of reduced cognitive ability in classical galactosemia and present evidence for an absence of substantial galactosemia-induced aggravation of this impairment with increasing age, at least in patients from 4 to 40 years of age. It remains to be clarified whether a reduction of cognitive function in galactosemia may be initiated by an in utero toxicity of endogenously formed galactose and which role such a process may play in the development of intellectual deficiencies that are later maintained throughout life.

On the mechanism ofl-alloisoleucine formation: Studies on a healthy subject and in fibroblasts from normals and patients with maple syrup urine disease

Summaryl-Alloisoleucine formation froml-isoleucine was studiedin vitro andin vivo. When a healthy subject was loaded withl-isoleucine, plasma levels ofl-isoleucine and 3-methyl-2-oxopentanoate (KMV), as well asl-alloisoleucine, increased. Peak values were reached successively and were in the orderl-isoleucine > > KMV > >l-alloisoleucine. Metabolic clearance ofl-isoleucine and KMV was rapid; clearance ofl-alloisoleucine was considerably delayed. When human skin fibroblast cultures were challenged withl-isoleucine, KMV accumulated at a gradually decreased rate, whereasl-alloisoleucine accumulated at a gradually accelerated rate. KMV andl-alloisoleucine formation were related and depended on thel-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 fibroblastsin vitro as opposed to the striking differences between healthy subjects and MSUD patientsin vivo are discussed with respect to the significance of physiological mechanisms participating in the formation and degradation ofl-alloisoleucine in man.

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-[1-13C]leucine bolus tests (38 μmol/kg body weight). Residual whole-body l-leucine oxidation was estimated on the basis of the 3-h kinetics of 13CO2 exhalation and 13C-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.

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 µmol/kg body weight per day in infants to about 0.2 µmol/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 µmol/kg body weight per day in infants to about 23 µmol/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.

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 indepen-dent 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≤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.

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

L-Alloisoleucine (2 S, 3 R), a diastereomer of L-isoleucine (2 S, 3 S), 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 μmol/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.

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.

Endogenous galactose formation in galactose-1-phosphate uridyltransferase deficiency

Abstract Patients with classical galactosaemia (galactose-1-phosphate uridyltransferase (GALT) deficiency) manifest clinical complications despite strict dietary galactose restriction. Therefore the significance of endogenous galactose production has been assessed. Previous in vivo studies primarily focused on patients homozygous for the most common genetic variant Q188R but little is known about other genetic variants. In the present study the endogenous galactose release in a group of non-Q188R homozygous galactosaemic patients (n = 17; 4–34 years) exhibiting comparably low residual GALT activity in red blood cells was investigated. Primed continuous infusion studies with D-[1-13C]galactose as substrate were conducted under post-absorptive conditions and in good metabolic control. The results demonstrate that all patients exhibiting residual GALT activity of <1.5% of control showed a comparable pathological pattern of increased endogenous galactose release irrespective of the underlying genetic variations. Possible implications of the findings towards a more differentiated dietary regimen in galactosaemia are discussed.

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

SummaryAssay 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 ⩾ 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 ⩾ 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.

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.