Mid-term outcome of 2 cases with maple syrup urine disease: role of liver transplantation in the treatment

BACKGROUND: Dietary treatment of maple syrup urine disease remains difficult; chronic nutritional support in the child does not always avoid acute crises so that liver transplantation may represent an alternate choice in some cases. CASE REPORTS: Two gypsy cousins were born by an interval of 4 days; both had maple syrup urine disease and were similarly treated from the first days of life. They were given exchange transfusions followed by diet restricted in the branched chain amino acids, maintaining normal growth and plasma leucine concentrations under 7 mg/100 ml. Laura, at 10 years, was retarded at school. Compliance to school attendance was limited by her diet problems. Helen suffered at 7 yr 3 mo from liver failure due to hepatitis A virus infection which required liver transplantation. Protein intake was normal 1 week later. At 10 years, she presented with the same degree of school retardation as her cousin, and was placed in the same class. CONCLUSION: Liver transplantation may be effective for treating metabolic problems in MSVD without significative difference between outcome post classic treatment or post liver transplantation.     

Diagnosis of a case of acute chloroquine poisoning using 1H NMR spectroscopy: characterisation of drug metabolites in urine

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO2 and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO2 and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H2O2 did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H2O2, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H2O2 also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H2O2 had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H2O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used.     

1H NMR spectroscopy studies of Huntington's disease: correlations with CAG repeat numbers

Huntington's disease (HD) is the result of an expanded (CAG) repeat in a gene on chromosome 4. A consequence of the gene defect may be progressive impairment of energy metabolism. We previously showed increased occipital cortex lactate in HD using localized 1H spectroscopy. We have now extended these studies to show an almost threefold elevation in occipital cortex lactate in 31 HD patients as compared with 17 normal control subjects (p < 10(-11)). The spectra in three presymptomatic gene-positive patients were identical to normal control subjects in cortical regions, but three in eight showed elevated lactate in the striatum. Similar to recently reported increases in task-related activation of the striatum in the dominant hemisphere, we found that striatal lactate levels in HD patients were markedly asymmetric (higher on the left side). Markers of neuronal degeneration, decreased N-acetylaspartate (NAA)/creatine and increased choline/creatine levels, were symmetric. Both decreased NAA and increased lactate in the striatum significantly correlated with duration of symptoms. When divided by his or her age, an individual's striatal NAA loss and lactate increase were found to directly correlate with the subject's CAG repeat number, with correlation coefficients of 0.8 and 0.7, respectively. Similar correlations were noted between postmortem cell loss and age versus CAG repeat length. Together, these data provide further evidence for an interaction between neuronal activation and a defect in energy metabolism in HD that may extend to presymptomatic subjects.     

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.     

NMR spectroscopy of human post mortem cerebrospinal fluid: distinction of Alzheimer''s disease from control using pattern recognition and statistics

Mutations of ras oncogenes in 37 human stomach cancers and 13 adenomas were investigated with regard to the histological phenotypes using polymerase chain reaction (PCR), allele-specific oligonucleotide hybridization and/or direct sequencing of the PCR products. The ras mutation was found only in one case (2.7%), the histology of which was poorly differentiated adenocarcinoma. We found no mutation in stomach adenomas. The mutation consisted of a guanine-to-adenine transition in the first base of codon 13 of c-Ki-ras which replaced wild-type glycine with serine, indicating that a putative glycine-to-aspartic acid change is not necessarily the critical event for c-Ki-ras gene activation in codon 13. These results further confirm the infrequency of ras mutation in stomach tumors and also suggest that ras mutations are not specific to the differentiated type of stomach cancer.     

Effects of replacement of Lys25 with Gln on the conformation of ribonuclease T1: sequence-specific 1H NMR resonance assignments of Gln25 ribonuclease T1 by two-dimensional NMR spectroscopy

Two isozymes of ribonuclease (RNase) T1 exist in nature, i.e. Gln25 RNase T1 and Lys25 RNase T1. Gln25 RNase T1 is less stable than Lys25 RNase T1, although the enzymatic activity is not distinguishable between these two isozymes. To elucidate the effects of the replacement of Lys25 with Gln on the conformation and microenvironments of RNase T1 in detail, two-dimensional NMR spectra were measured, sequence-specific 1H NMR resonance assignments of Gln25 RNase T1 were performed, and then the determined parameters and microenvironments of Gln25 RNase T1 were compared with those of Lys25 isozyme [Hoffmann, E. and Rüterjans, H. (1988) Eur. J. Biochem. 177, 539-560]. The main chain protons were assigned for 101 out of the total of 104 amino acid residues. Secondary structure elements were identified from analysis of characteristic NOE patterns, interstrand NOE connectivities, and hydrogen-deuterium exchange rates of main chain amide protons. The results indicated that Gln25 RNase T1 contains a single alpha-helix and seven beta-strands. The secondary structure of Gln25 RNase T1 is, thus, essentially the same as that of Lys25 RNase T1. On the other hand, comparison of the conformation-dependent shifts of Gln25 RNase T1 with these of Lys25 RNase T1 showed that the replacement of Lys25 with Gln has significant effects on the C-terminal part of the alpha-helix region and the base-binding site. These results may indicate that the base-binding site is relatively flexible in the RNase T1 molecule. Among the residues of the C-terminal part of the alpha-helix region, the protons of Asp29 were most affected in terms of their chemical shifts, which may indicate that the side chain carboxylate anion of Asp29 is the counterpart of the electrostatic interaction of Lys25 in Lys25 RNase T1. The Gln25 of Gln25 RNase T1 may have little or no interaction with Asp29, and this may be the reason why Gln25 RNase T1 is less stable than the Lys25 isozyme.     

Effect of anaemia correction on skeletal muscle metabolism in patients with end-stage renal disease: 31P magnetic resonance spectroscopy assessment

Skeletal muscle metabolism during exercise was compared in 5 patients with end-stage renal disease (ESRD) and 8 healthy controls, using a noninvasive technique, 31P magnetic resonance spectroscopy (MRS). After 3 months of anaemia correction with recombinant human erythropoietin (rHuEPO) these patients were re-evaluated. Maximal power achieved by the ESRD patients during a dynamic wrist flexion exercise test was 33% lower (p < 0.05) than the controls. Similarly in the ESRD group, the power at the onset of metabolic acidosis (the intracellular threshold) was 29% less than controls. The metabolic differences observed in the patients indicated a lower aerobic capacity. Three months of rHuEPO treatment resulted in a 55% increase in mean haematocrit but conferred no significant improvement in metabolic parameters at rest or during exercise. The lack of any significant changes in muscle metabolism following the correction of anaemia suggests that oxygen availability is not the exclusive limiting factor for aerobic metabolism in ESRD patients.     

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.     

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.