Mutation and DNA modification in Salmonella exposed to N-nitrosodimethylamine under UVA- and sunlight-irradiation.

Previously, we reported that when Salmonella typhimurium and Escherichia coli were treated with N-nitrosodimethylamine (NDMA) under irradiation with ultraviolet-A (UVA), mutagenesis of the bacteria took place without externally added activation enzymes. We also observed the formation of O(6)-methylguanine (O(6)-meG), N(7)-methylguanine (N(7)-meG) and 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) in calf thymus DNA treated with NDMA plus UVA. In this study, we observed the mutagenicity of NDMA under irradiation of natural sunlight in S. typhimurium. Furthermore, we detected the formation of O(6)-meG, N(7)-meG and 8-oxodG in calf thymus DNA treated with NDMA plus simulated sunlight. Regarding the mutagenesis of S. typhimurium by NDMA plus UVA, we have now identified and quantified O(6)-meG formed in the genomic DNA of the bacteria under conditions of the mutagenesis.

Characterization of azo coupling adducts of benzenediazonium ions with aromatic amino acids in peptides and proteins.

A synthetic peptide, VLSPADKTNWGHEYRMF(cmC)QIG, was reacted with 4-chlorobenzenediazonium hexafluorophosphate as a model for reactions of aromatic diazonium ions with proteins. At a ratio of diazonium ion to peptide of 0.8:1, three products could be seen by reversed-phase HPLC. Electrospray mass spectrometric analysis of the isolated products revealed that two of the products had the same mass of 2648 Da, being 138 Da higher than the parent peptide and corresponding to the addition of a 4-chlorobenzenediazo group. The third isolated product had a mass of 2787 Da which corresponded to the addition of two 4-chlorobenzenediazo groups (276 Da). Digestion of the monoadducted intact peptides with trypsin or endoproteinase Glu-C and HPLC separation of adduct oligopeptides followed by sequencing with electrospray ionization tandem mass spectrometry showed unambiguously that histidine and tyrosine residues were the major sites of modification. Incubation of human serum albumin with 4-chlorobenzenediazonium hexafluorophosphate at molar ratios of 1:1, 1:2, and 1:10 resulted in adduct formation as detected by shifts in the HPLC retention time of the protein and also by an increase in mass as determined by electrospray mass spectrometry.

Subunit molecular mass assignment of 14,654 Da to the soluble beta-galactoside-binding lectin from bovine heart muscle and demonstration of intramolecular disulfide bonding associated with oxidative inactivation.

The soluble dimeric beta-galactoside-binding lectin (subunit molecular mass, approximately 14 kDa) of bovine heart muscle, in common with the 14-kDa lectins of several other animal species, displays carbohydrate-binding activity when it is in the reduced state, but the purified lectin loses this activity upon oxidation. In the present study, the presence of any post-translational modification and the mechanism of the oxidative inactivation have been investigated by analyses of the reduced and oxidized forms of the purified bovine lectin by electrospray ionization-mass spectrometry (ESI-MS) and by liquid secondary ion mass spectrometry (LSIMS) of tryptic and peptic peptides. By ESI-MS, the molecular mass of the reduced lectin is determined to be 14,654.6 +/- 0.9 Da, and that of the oxidized lectin is 14,649.3 +/- 1.1 Da. These masses correspond to the amino acid sequence of the protein with the cysteines having free sulfhydryl groups in the reduced state and forming disulfide bonds in the oxidized state. There is no evidence of post-translational modification in either lectin form except for monoacetylation already predicted for alanine at the blocked N-terminal end. Pronounced differences in charge distribution in the electrospray ionization mass spectra of the reduced and oxidized lectin, reflecting a change in the number of accessible protonation sites in the oxidized protein, are consistent with the protein being held in an altered conformation by covalent bonding. The results of LSIMS analyses of tryptic and peptic peptides in conjunction with Edman sequencing indicate that disulfide bonding occurs predominantly between Cys2 and Cys130, Cys16 and Cys88, and Cys42 and Cys60. There is no evidence of oxidation of Trp68. These results, taken together with observations that almost the complete polypeptide chain is necessary for the functional integrity of the carbohydrate recognition domain (Abbott, W. M., and Feizi, T. (1991) J. Biol. Chem. 266, 5552-5557) point to intramolecular disulfide bonding with a change in protein folding and conformation as the mechanism of oxidative inactivation of the purified bovine lectin.

Enzymologic studies on patients with methylmalonic aciduria: basis for a clinical trial of deoxyadenosylcobalamin in a hydroxocobalamin-unresponsive patient.

Eleven patients with methylmalonic aciduria have been classified on the basis of detailed enzymology on cultured skin fibroblasts. Nine were classified as mutase deficiencies and were unresponsive to hydroxocobalamin in vivo or in vitro. One was classified as a Cbl A variant and was responsive to hydroxocobalamin therapy in vitro and in vivo. Patient 11 was classified as having deoxyadenosyltransferase deficiency (Cbl B). However, a clinical therapeutic trial of deoxyadenosylcobalamin resulted in no clinical or biochemical improvement. Further studies on the patient's cultured fibroblasts suggested that deoxyadenosylcobalamin fails to reach the mitochondria in an intact form. These studies show that detailed enzymologic classification is essential for the reliable evaluation of the response to therapeutic maneuvers; complementation studies alone may be inadequate to completely classify these patients. Therapy with deoxyadenosylcobalamin offers no advantages over the use of hydroxocobalamin in the treatment of patients with methylmalonic aciduria.

Prenatal diagnosis of 3-hydroxy-3-methylglutaric aciduria by GC-MS and enzymology on cultured amniocytes and chorionic villi.

This paper reports the prenatal diagnosis of HMG CoA lyase deficiency at 16 weeks' gestation by direct chemical analysis of cell-free amniotic fluid and by measurement of HMG CoA lyase activity in cultured amniocytes. Termination of an affected fetus allowed study of chorionic villus tissue, the results providing the basis for future first trimester prenatal diagnoses of this condition. An abstract report of this work has appeared elsewhere.

Urinary C6-C12 dicarboxylic acylcarnitines in Reye's syndrome.

C6-C12 dicarboxylic acylcarnitines have been identified for the first time in urine from a 2-year-old girl presenting with Reye's syndrome. The acylcarnitines were extracted by ion-exchange chromatography and analysed, both underivatised and as methyl esters using high-resolution fast-atom-bombardment mass spectrometry and B/E-linked scanning. The acylcarnitines were quantified by capillary gas chromatography of the acids extracted after hydrolysis of the acylcarnitine esters. Dodecandioylcarnitine was present in the highest concentration (35.9 mmol/mol creatinine) which exceeded the urinary free dodecandioic acid concentration. The adipic, suberic and sebacic acylcarnitine concentrations were less than 10% of the respective free acid concentrations. It is possible that beta-oxidation of dicarboxylic acids is partially inhibited in Reye's syndrome leading to accumulation of precursor dodecandioyl CoA which is metabolised to dodecandioylcarnitine. The accumulation of these metabolic intermediates may be significant in the pathogenesis of Reye's syndrome.

Contribution of gut bacterial metabolism to human metabolic disease.

Metronidazole, an antibiotic with specific activity against anaerobic bacteria, was of clinical and biochemical benefit in two patients with methylmalonic aciduria. The virtual elimination of propionic acid from the stool suggested that propionic acid derived from faecal bacterial metabolism contributes substantially to methylmalonate production. These findings point to a novel avenue of treatment for these disorders of intermediary metabolism, and indicate the importance of microbial gut flora in normal human metabolism.

The response to L-carnitine and glycine therapy in isovaleric acidaemia.

The profound metabolic disturbances which occur in isovaleric acidaemia are due to the intramitochondrial accumulation of isovaleryl coenzyme A (CoA) with a consequent reduction in the availability of free CoA. Secondary carnitine insufficiency is also a feature of this and other disorders of organic acid metabolism. A patient who presented at 2.5 years of age was diagnosed using capillary GC-MS as having isovaleric acidaemia. She showed the full spectrum of abnormal organic acids previously associated with the 'neonatal' form of the disease despite her late presentation, indicating that it is inappropriate to refer to acute early and late onset forms of isovaleric acidaemia. Instead, a spectrum of disease exists, determined by environmental factors, residual enzyme activities and modifying effects of different phenotypes in different individuals. She also showed evidence of carnitine insufficiency. An oral challenge with L-carnitine resulted in the excretion of large amounts of urinary acylcarnitines which were shown by use of fast atom bombardment mass spectrometry to be primarily isovalerylcarnitine. Regular glycine supplementation caused no significant increase in urinary isovalerylglycine and had to be stopped because of side-effects after 5 days. An oral L-carnitine challenge during glycine supplementation resulted in a marked increase in isovalerylglycine excretion, again associated with the excretion of large amounts of isovalerylcarnitine. Carnitine acts by removing (detoxifying) intramitochondrial isovaleryl groups and, in the presence of glycine, it promotes the formation of isovalerylglycine. We believe L-carnitine supplementation is of value in the treatment of isovaleric acidaemia and that, in the present case, L-carnitine together with a moderate dietary restriction has proved to be the optimum form of therapy.

Dizygotic twins with 3-hydroxy-3-methylglutaric aciduria; unusual presentation, family studies and dietary management.

A 4-month-old infant with hypotonia and macrocephaly was diagnosed as having 3-hydroxy-3-methylglutaric aciduria, using gas chromatography and mass spectrometry and confirmatory enzyme studies. The same diagnosis was made on his asymptomatic non-identical twin. Examination of the pedigree is consistent with an autosomal recessive mode of inheritance. Dietary treatment improved the symptoms of the propositus, but did not prevent episodes similar to Reye's syndrome in both twins. One such episode closely followed immunisation and our experience suggests that children with this disorder should be observed carefully following immunisation. These episodes were accompanied by an overflow of a wide range of abnormal metabolites. Examination of the urine for organic acids should be considered in infants with unexplained hypotonia and macrocephaly, especially if accompanied by abnormal biochemical indices.

Metabolic response to carnitine in methylmalonic aciduria. An effective strategy for elimination of propionyl groups.

Patients with methylmalonic aciduria have an excessive intramitochondrial accumulation of acylcoenzyme A compounds that may reduce the availability of free coenzyme A (CoA) for normal metabolic requirements, producing profound metabolic disturbances. Giving carnitine to a patient with methylmalonic aciduria produced an increase in hippurate excretion (an index of intramitochondrial adenosine triphosphate (ATP) and CoA availability), a large increase in short chain urinary acylcarnitines, and a reduction in excretion of methylmalonate and methylcitrate. These acylcarnitines were shown by fast atom bombardment and B/E linked scan mass spectrometry to be propionylcarnitine and acetylcarnitine. Carnitine acts by removing (detoxifying) propionyl groups, thereby releasing CoA and restoring ATP biosynthesis and concentrations towards normal. L-carnitine may play a central role in maintenance of mitochondrial and cellular homoeostasis in methylmalonic aciduria and propionic acidaemia. These principles may provide an approach to the treatment of this and other disorders, inherited and acquired, in which accumulation of acyl CoA metabolites results in sequestration of free CoA, thereby perturbing metabolic homoeostasis.

Urinary organic acids in normal full-term newborns aged 1-7 days.

urinary organic acid metabolite excretion patterns in normal newborns aged 1 to 7 days were analysed by GC and MS. Statistical analysis showed significantly different relationships between metabolite excretion, age, sex and feed. The data provide a necessary background for the diagnosis of inborn errors or organic acid metabolism.