Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water.

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.

Pitfalls in the use of 2-octynoic acid as an in vivo model of medium-chain acyl-coenzyme A dehydrogenase deficiency: ketone turnover and metabolite studies in the rat.

2-Octynoic acid was administered by intraperitoneal injection to fasted Sprague-Dawley rats in an attempt to simulate medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. The resultant urine organic acid profile showed a mild dicarboxylic aciduria but lacked the glycine conjugates characteristic of MCAD deficiency. Further studies with infused 13C(4)-3-hydroxybutyrate and 13C(2)-acetoacetate demonstrated reduced ketone production in treated rats compared with control animals. Although plasma ketone body concentrations were low in treated rats, plasma free fatty acids were also low, thereby providing diminished substrate for ketone production. This is the reverse of the finding in children with MCAD deficiency, who have low levels of plasma ketones despite elevated free fatty acids. These animal studies were therefore not helpful in improving our understanding of ketone body kinetics in children with MCAD deficiency.

Fasting medium chain acyl-coenzyme A dehydrogenase--deficient children can make ketones.

Medium chain acyl-coenzyme A dehydrogenase (MCAD) deficiency classically presents as hypoketotic hypoglycemia. Under-production of ketones has been presumed to be the cause of hypoketosis, but this has never been proven. Stable isotope dilution studies of ketone kinetics were performed on three well children with homozygous 985G MCAD deficiency using 1,3-13C2 sodium acetoacetate and 1,2,3,4-13C4 sodium 3-hydroxybutyrate to ascertain the rates of ketone production, interconversion, and use. All children were fasted for 9 to 11.5 hours before the beginning of the study period. Euglycemia was maintained in all cases. Ketone kinetics were calculated using a two-accessible pool model and showed normal ketone production in all three children compared with published control data from children fasted for a similar length of time. There is no evidence for underproduction or overuse of ketones in these MCAD-deficient children, at least when they are well. We propose that another factor, such as fever, may be required to reduce ketone production and result in the biochemical phenotype recognized in unwell children.

Excretion of 3,6-epoxydicarboxylic acids in peroxisomal disorders.

The urinary excretions of several organic acids were quantitatively studied by gas chromatography/mass spectrometry in subjects with disorders of peroxisome biogenesis (n = 8) and controls (n = 26). The excretion of 3,6-epoxtetradecanedioic acid was significantly elevated in all subjects with disorders of peroxisome biogenesis (1.8-20.8; controls, not detected-0.5, mumol/mmol of creatinine). 3,6-Epoxydodecanedioic acid excretion was usually elevated (1.4-19.8; controls, not detected-4.2) and 3,6-epoxyoctanedioic acid excretion was not elevated not detected-8.8; controls, 0.6-9.5 mumol/mmol of creatinine). It is suggested that measurement of 3,6-epoxydicarboxylic acids may be useful for the diagnosis of these disorders.

VLCAD deficiency: pitfalls in newborn screening and confirmation of diagnosis by mutation analysis.

We diagnosed six newborn babies with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) through newborn screening in three years in Victoria (prevalence rate: 1:31,500). We identified seven known and two new mutations in our patients (2/6 homozygotes; 4/6 compound heterozygotes). Blood samples taken at age 48-72 h were diagnostic whereas repeat samples at an older age were normal in 4/6 babies. Urine analysis was normal in 5/5. We conclude that the timing of blood sampling for newborn screening is important and that it is important to perform mutation analysis to avoid false-negative diagnoses of VLCADD in asymptomatic newborn babies. In view of the emerging genotype-phenotype correlation in this disorder, the information derived from mutational analysis can be helpful in designing the appropriate follow-up and therapeutic regime for these patients.

Novel glycine conjugates in medium-chain acyl-CoA dehydrogenase deficiency.

The glycine conjugates of isocaproic, 4-methylhexanoic, 7-hydroxyoctanoic and 8-hydroxyoctanoic acids have been identified in the urine of children with medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using gas chromatography-mass spectrometry of the trimethylsilyl derivatives. A quantitative study showed that the glycine conjugates of isocaproic and 4-methylhexanoics acids were excreted during acute episodes and in smaller amounts when subjects were asymptomatic. The glycine conjugates of 7-hydroxyoctanoic and 8-hydroxyoctanoic acids were detectable during acute episodes. None of the conjugates was detected in controls or controls receiving a diet containing medium-chain triglycerides. It is suggested that the glycine conjugates of isocaproic acid and 4-methylhexanoic acid are metabolites of branched-chain fatty acids and that they are specific for MCAD deficiency.

Oligosaccharide characterization and quantitation using 1-phenyl-3-methyl-5-pyrazolone derivatization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of monosaccharides, maltooligosaccharides, and oligosaccharides enzymatically released from asparagine-linked sites in ribonuclease B and fetuin have been investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Use of the matrix 2,6-dihydroxyacetophenone containing diammonium hydrogen citrate (DHAP/DAHC) resulted in predominance of protonated over sodiated pseudomolecular ions of PMP-derivatized oligosaccharides. By comparison, the matrices alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid resulted in predominantly sodiated pseudomolecular ions. In addition, tendencies for fragmentation of PMP-oligosaccharide derivatives were significantly lower with DHAP/DAHC which enabled meaningful data to be obtained in reflector mode, even for samples with high excipient levels. The relative magnitude of the ion signals for PMP-derivatized maltooligosaccharides and ribonuclease B oligosaccharides correlated well with the oligomer distribution apparent by HPLC. PMP-maltohexose was used as an internal standard to quantitate PMP-oligosaccharides from ribonuclease B and asialofetuin in crude derivatization mixtures. A linear relationship was observed between the ratio of the intensities of pseudomolecualr ions and the amount of glycoprotein derivatized. The limit of detection for the major oligosaccharide of each protein was reached with ca. 3 micrograms of glycoprotein but may be further enhanced by optimization of sample handling. PMP derivatives of sialylated fetuin oligosaccharides were readily detected as protonated pseudomolecular ions by linear mode analyses. By comparison, reflector mode analyses revealed substantially reduced magnitudes of protonated pseudomolecular ions and considerable post-source fragmentation of sialic acid residues. The PMP derivatives of fetuin oligosaccharides were also amenable to exoglycosidase treatment as shown by the mass shifts found upon treatment with sialidase.

Transient 5-oxoprolinuria and high anion gap metabolic acidosis: clinical and biochemical findings in eleven subjects.

We describe biochemical and clinical features of 11 subjects (ages, 1.2-84 years, nine females and two males) with transient 5-oxoprolinuria (0.6-23.6 mol/mol of creatinine, reference range <0.07). A variety of conditions preceded the onset of acidosis, and all had taken acetaminophen (paracetamol), although in therapeutic amounts in most subjects. Metabolic acidosis was documented in nine subjects, and all had an increased anion gap and abnormal liver functions. 5-Oxoproline was the major urinary organic acid in five subjects, whereas the rest had more complex profiles comprising 5-oxoproline and other organic acids, such as lactate, 3-hydroxybutyrate, and 4-hydroxyphenyl lactate. The 5-oxoproline was predominantly of the L-configuration. One subject died during an acidotic episode, and the rest recovered with no apparent long-term ill effects. Urinary 5-oxoproline was within the reference range in six subjects that were re-tested after the anion gap normalized. These findings suggest that acetaminophen, in association with other unidentified factors, is involved in the development of this condition through a mechanism of depletion of liver glutathione stores.

Pseudo-glutarylcarnitinaemia in medium-chain acyl-CoA dehydrogenase deficiency detected by tandem mass spectrometry newborn screening.

As well as characteristic increases in C(8) carnitine, dried blood spot samples from 11 newborns with medium-chain acyl-CoA dehydrogenase deficiency detected by tandem mass spectrometry screening using butyl esters showed apparent increases in glutarylcarnitine (m / z 388 signals). In four of the newborns in which it was measured, apparent increases in malonylcarnitine (m / z 360) were also detected. It was shown that the apparent increases were caused by interfering acylcarnitines, putatively identified as hydroxyoctanoylcarnitine and hydroxydecanoylcarnitine, respectively, using alternative derivatives for tandem mass spectrometry. Levels of the two abnormal carnitines correlated with C(8) carnitine levels and normalized with repeat testing in 10 cases. These results indicated that the abnormal carnitines were significantly elevated only during periods of increased fatty acid catabolism, as may occur in the immediate postnatal period.

Determination of the disulfide bond arrangement of Newcastle disease virus hemagglutinin neuraminidase. Correlation with a beta-sheet propeller structural fold predicted for paramyxoviridae attachment proteins.

Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Until this study, the disulfide linkages of this HN and structurally similar attachment proteins of other members of the paramyxoviridae family were undefined. To define these linkages, disulfide-linked peptides were produced by peptic digestion of purified HN ectodomains of the Queensland strain of Newcastle disease virus, isolated by reverse phase high performance liquid chromatography, and analyzed by mass spectrometry. Analysis of peptides containing a single disulfide bond revealed Cys(531)-Cys(542) and Cys(172)-Cys(196) linkages and that HN ectodomains dimerize via Cys(123). Another peptide, with a chain containing Cys(186) linked to a chain containing Cys(238), Cys(247), and Cys(251), was cleaved at Met(249) with cyanogen bromide. Subsequent tandem mass spectrometry established Cys(186)-Cys(247) and Cys(238)-Cys(251) linkages. A glycopeptide with a chain containing Cys(344) linked to a chain containing Cys(455), Cys(461), and Cys(465) was treated sequentially with peptide-N-glycosidase F and trypsin. Further treatment of this peptide by one round of manual Edman degradation or tandem mass spectrometry established Cys(344)-Cys(461) and Cys(455)-Cys(465) linkages. These data, establishing the disulfide linkages of all thirteen cysteines of this protein, are consistent with published predictions that the paramyxoviridae HN forms a beta-propeller structural fold.

Trimethylaminuria, fish odour syndrome: a new method of detection and response to treatment with metronidazole.

Trimethylaminuria is an autosomal recessive disorder involving deficient N-oxidation of the dietary-derived amine trimethylamine (TMA). TMA, a volatile tertiary amine, accumulates and is excreted in urine of patients with deficient TMA oxidase activity. Treatment strategies for this condition are limited. We report a new stable-isotope dilution method for rapid sequential analysis of TMA concentrations and the clinical and biochemical response to treatment with metronidazole.

Succinic semialdehyde dehydrogenase deficiency: low excretion of metabolites in a neonate.

A neonate at risk for succinic semialdehyde dehydrogenase deficiency was investigated on day 1. The urine level of 4-hydroxybutyrate was only slightly elevated (23 mumol/mmol of creatinine; controls 1.6-14, n = 18). This value was considerably less than those found for older children with succinic semialdehyde dehydrogenase deficiency and made interpretation of the result uncertain. The diagnosis of succinic semialdehyde dehydrogenase deficiency was confirmed by enzyme assay, and repeat urine testing showed a steady increase in the level of 4-hydroxybutyrate to 359 mumol/mmol at 6 months.

Diagnosis of 21-hydroxylase deficiency in newborn infants by GC-MS of urinary steroids.

In a study using gas chromatography-mass spectrometry (GC-MS) on urine specimens from 16 normal infants and 16 infants with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (aged 1 day to 4 weeks), the major steroids recognized in all infants were: 16 alpha-hydroxy-dehydroepiandrosterone, 16 beta-hydroxy-dehydroepiandrosterone, 16-oxo-androstenediol, androstenetriol, 15 beta,17 alpha-dihydroxy-pregnenolone and 16 alpha-hydroxy-pregnenolone. Pregnanetriol was detectable in three normal infants (aged 3, 6 and 15 days) but the levels seen in 15 CAH patients were in a higher range. Pregnanetriolone, 5 beta-17-hydroxy-pregnanolone and 15 beta,17 alpha-dihydroxy-pregnanolone were present in the urine of 15 CAH patients, but were not detectable in any of the normal infants. The older the patient, the higher the level was of each of these four steroids. The results indicate that, even on day 1, patients with CAH due to 21-hydroxylase deficiency may be positively identified using GC-MS of urine specimens. This does not preclude the possibility that a minority of patients with CAH, most likely those with mild 21-hydroxylase deficiency, may not exhibit the characteristic GC-MS findings on day 1, as seen in one of the 16 CAH patients.

Episodes of severe metabolic acidosis in a patient with 3-methylglutaconic aciduria.

Persistent excretion of 3-methylglutaconic acid was found in a 6-month-old infant with multiple minor physical malformations and delayed development. During two episodes of intercurrent viral illness, the patient developed severe metabolic acidosis and excreted large amounts of lactate, 3-hydroxybutyrate and acetoacetate. The excretion of 3-methylglutaconic acid did not change during these episodes, nor did it increase following leucine loading. In vitro studies suggest that in this patient, as in the majority of other patients with 3-methylglutaconic aciduria, a primary defect in leucine metabolism is not responsible for the biochemical abnormality.

Striatal degeneration in glutaric acidaemia type II.

A girl of first cousin parents presented in the 1st year of life with a progressive neurological disease with muscle weakness and hypotonia, accompanied later by dystonia. Investigations, including gas chromatography of urine, showed no abnormality. Autopsy showed marked neuronal loss and gliosis in the putamen and globus pallidus. The activity of glutaryl-CoA dehydrogenase in cultured fibroblasts was normal, but the activity of electron transfer flavoprotein was markedly diminished. Retrospective study of urine by capillary gas chromatography/mass spectrometry showed small amounts of glutaric and other organic acids. This is the first report of striatal degeneration in association with glutaric acidaemia type II. The neuropathological changes were milder than those in glutaric acidaemia type I.

In vivo disposal of phenylalanine in phenylketonuria: a study of two siblings.

Mutation at the phenylalanine hydroxylase (PAH) locus is a cause of hyperphenylalaninaemia. Genotype-phenotype correlation relative to the predicted PAH activity may differ at the metabolite level and at the IQ level in untreated phenylketonuria. Discordant metabolic phenotypes have been noted in siblings; influences on transport and metabolism of phenylalanine determining homeostasis may account for differing metabolic phenotypes. We report two siblings of different sex and identical genotype at the PAH locus who demonstrate a difference in phenylalanine disposal. A stable isotope infusion of [2H5]phenylalanine was used to measure protein turnover, phenylalanine hydroxylation and excretion of phenylalanine transamination metabolites. The siblings were observed to have identical hydroxylation rates under the experimental conditions of the study while manifesting differences in renal excretion rates of phenylalanine transamination metabolites and protein accretion.