[Clinical phenotypic and genotypic analysis of 5 pediatric patients with ß-ketothiolase deficiency].

Objective: To summarize the clinical and genetic characteristics of children with ß-ketothiolase deficiency (BKTD). Methods: The clinical characteristics, biochemical, markers detected by tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC/MS), as well as the variants in ACAT1 gene among 5 children with BKTD in Children's Hospital of Chongqing Medical University between October 2018 and December 2022 were retrospectively analyzed. Results: The onset age of the disease in 5 patients (4 males and 1 female) ranged from 9.7 to 28.0 months. During the acute phase, severe metabolic acidosis was observed with a pH of 6.9-7.1, as well as hypoglycaemia (2.3-3.4 mmol/L) and positive urinary ketone bodies (+-++++). Blood levels of methylcrotonyl carnitine, methylmalonyl carnitine and malonyl carnitine were 0.03-0.42, 0.34-1.43 and 0.83-3.53 µmol/L respectively and were significantly elevated. Urinary 2-methyl-3-hydroxybutyric acid was 22-202 and 3-hydroxybutyric acid was 4-6 066, both were higher than the normal levels. Methylcrotonylglycine was mild elevated (0-29). The metabolites detected by MS/MS and GC/MS were significantly reduced after treatment. Analysis of ACAT1 gene mutation was performed in 5 children. Most variants were missense (8/9). Four previously unreported variants were identified: c.678G>T (p.Trp226Cys), c.302A>G (p.Gln101Arg), c.627_629dupTGA (p.Asn209_Glu210insAsp) and c.316C>T (p.Gln106Ter), the first 2 variants were predicted to be damaging by SIFT, PolyPhen-2 and Mutation Taster software. c.316C>T (p.Gln106Ter) is a nonsense variant. Conclusions: ß-ketothiolase deficiency is relatively rare, lacks specific clinical manifestations, however severe metabolic acidosis, hypoglycemia, and ketosis during the acute onset were consistent findings. Missense mutations in the ACAT1 gene are common genetic causes of ß-ketothiolase deficiency.

[The pH-Sensitive Potassium Channel TASK-1 Is a Chemosensor for Central Respiratory Regulation in Rats].

TWIK-related acid-sensitive potassium channel-1 (TASK-1) is a "leak" potassium channel sensitive to extracellular protons. It contributes to setting the resting potential in mammalian neurons. TASK-1 channels are widely expressed in respiratory-related neurons in the central nervous system. Inhibition of TASK-1 by extracellular acidosis can depolarize and increase the excitability of these cells. Here we describe the distribution of TASK-1 in the rat brainstem and show that TASK-1 mRNAs are present in respiratory-related nuclei in the ventrolateral medulla, which have been proposed as neural substrates for central chemo-reception in rats. After inhalation of 8% CO2 for 30 and 60 min, TASK-1 mRNA levels in positive-expression neurons were remarkably upregulated. Injection of the TASK-1 blocker anandamide (AEA) into the rat lateral cerebral ventricle, showed a significant excitement of respiratory at 10 min posttreatment, with a marked decrease in inspiratory and expiratory durations and an increased frequency of respiration. We suggest that TASK-1 channel may serve as a chemosensor for in central respiration and may contribute to pH-sensitive respiratory effects. TASK-1 channel might be an attractive candidate for sensing H^(+)/CO2 in several respiratory-related nuclei in the brainstem. It is likely that TASK-1 participates in pH-sensitive chemical regulation in the respiratory center under physiological and pathological conditions.

Fragmentation mechanisms of oligodeoxynucleotides: effects of replacing phosphates with methylphosphonates and thymines with other bases in T-rich sequences.

This article reports another step in an ongoing effort to understand the fragmentation of T-rich oligodeoxynucleotides. We extended an earlier investigation of T-rich 4-mers to T-rich 6-mers, 8-mers and 10-mers by using four different tandem mass spectrometric methods. The methods include low-energy collisionally activated decomposition (CAD) of electrospray ionization (ESI)-produced ions, source-CAD of ESI-produced ions, post-source decay (PSD), and CAD of matrix assisted laser desorption ionization (MALDI)-generated ions. The most abundant fragment ions produced from [M - 2H]2- precursors upon low-energy CAD in an ion trap are the [a - B]- and their complementary w ions. The predominant cleavage sites for T-rich oligodeoxynucleotides are always the 3' C-O bonds adjoining a non-T nucleobase (i.e., a base with a higher proton affinity (PA) than that of T). The relative abundance of [a - B]- correlates with the PAs of the nucleobases, underscoring the importance of proton transfer to the base. The propensity to form [a - B]- ions falls in the order of G > C approximately A >> T. Structural isomers up to 10-mers can be readily sequenced and distinguished with each of the four tandem mass spectrometric methods applied. The fragmentation of oligodeoxynucleotides in which various phosphates were replaced with methylphosphonate is a measure of the participation of the phosphate proton in the formation of [a - B] ions. For 4 and 5-mers, transfer of an acidic proton from the 5'-phosphate to the departing base is the initiating step in the formation of [a - B]- ions.

Metastable decay of negatively charged oligodeoxynucleotides analyzed with ultraviolet matrix-assisted laser desorption/ionization post-source decay and deuterium exchange.

In an effort to understand the initiating step in metastable-ion decay of UV matrix-assisted laser desorption/ionization (MALDI)-produced ions, we conducted experiments in which we exchanged all active protons for deuterons of tetrameric and hexameric oligodeoxynucleotides. We wish to address the controversial proposal that in the negative-ion mode, as in the positive-ion mode, fragmentation is driven by nucleobase protonation. The results show unambiguously that proton transfer, leading to zwitterion formation, charges a nucleobase prior to its elimination. The zwitterion formation directs fragmentation of both positive and negative oligodeoxynucleotide ions. Poly-T-rich oligodeoxynucleotide tetramers show surprising differences in the negative compared to the positive-ion mode, as thymine is preferentially expelled, instead of a nucleobase with higher proton affinity. For the exceptional case of negatively charged poly-T-rich oligodeoxynucleotide tetramers generated by MALDI, we propose that zwitterion formation with positive charging of a nucleobase leads to base stabilization in the negative-ion mode through an interaction of the positive nucleobase with the excess negative charge. Moreover, backbone cleavages (accompanied by H rearrangement) of a phosophodiester bond give first-generation products that can be traced back to this tripolar complex bearing one positive and two negative charges, all of which may be interacting.

Fragmentation mechanisms of oligodeoxynucleotides studied by H/D exchange and electrospray ionization tandem mass spectrometry.

We used solution-phase hydrogen/deuterium (H/D) exchange and multistage tandem mass spectrometry (MS/MS) experiments in an electrospray ion-trap mass spectrometer operating in the negative-ion mode to investigate the consequences of the loss of a high proton-affinity (PA) base from T-rich tetra and hexadeoxynucleotides. The T-rich oligodeoxynucleotides containing one or two other nucleobases take advantage of the mass spectral inertness of T because fragmentation of a T-rich oligomer is simple, allowing a tight focus on those processes of interest. Furthermore, determination of T-rich oligodeoxynucleotides may be a starting point in the development of a mass spectrometric scheme to understand the mutagenicity of various types of DNA damage by UV radiation. For nine oligodeoxynucleotides, the nucleobases were charged by nearly exclusive D transfer and then expelled as neutral bases. Loss of the base located at the 3' end is preferred over that from the 5' terminus when the two bases are identical. The observation of partially exchanged fragments from a completely exchanged precursor ion proves intramolecular H/D exchange between hydrogen atoms that can exchange in water and those that cannot. The multiplicity of the product-ion peaks provides information on decomposition pathways and origins of the product ions and shows that the loss of base is the first step in all fragmentation of hexanucleotides, but is a competitive process for tetranucleotide fragmentation.

Gas-phase stability of double-stranded oligodeoxynucleotides and their noncovalent complexes with DNA-binding drugs as revealed by collisional activation in an ion trap.

The intrinsic (gas-phase) stabilities of duplex, self-complementary oligonucleotides were measured in a relative way by subjecting the duplex precursor ions to increasing amounts of collision energy during the collisional-activated decomposition (CAD) events in an ion-trap mass spectrometer. The results are displayed as a dissociation profile, an s-shaped curve that shows the dependence of the relative abundance of the duplex on the applied collision energy. The total number of charges, the total number of base pairs, and the location of the high proton-affinity bases (i.e., G and C) are the main factors that affect the intrinsic stability of the duplex oligonucleotides. If the charge state is the same, the stability, as measured as a half-wave collision energy, E1/2, correlates well with the total number of H bonds for the duplex. The intrinsic stabilities of noncovalent complexes between duplex oligonucleotide and some DNA-binding drugs were also measured by using the newly developed method. Although duplexes are stabilized in the gas phase when they bind to drug molecules, correlations between gas-phase stabilities and the solution-binding affinities have not yet been obtained. Complexes in which the drug is bound in the minor groove must be joined tightly because they tend to dissociate in the gas phase by breaking covalent bonds of the oligonucleotide to give base loss and small sequence-ion formation. Complexes in which the drug is known to favor intercalation dissociate by breaking weak, noncovalent bonds to form single-stranded oligonucleotides although cleavage of covalent bonds of the oligonucleotide also occurs.

Structure and fragmentation mechanisms of isomeric T-rich oligodeoxynucleotides: a comparison of four tandem mass spectrometric methods.

Understanding the product-ion spectra of T-rich tetradeoxynucleotides is a starting point in the development of a mass spectrometric scheme to determine the mutagenicity of individual types of DNA damage. We obtained product-ion spectra for electrospray-produced ions that were activated in the ion source (electrospray ionization-source collision-activated-dissociation) and by high-energy collisions in the MS/MS mode of a four-sector instrument. We also activated singly and doubly charged ions by low-energy collisions in an ion-trap mass spectrometer and investigated post source decompositions of matrix-assisted laser desorbed ions in a time-of-flight mass spectrometer. The various methods of extracting structural information give remarkably consistent results. The difference in the relative abundances of wn and dn ions of the singly charged oligonucleotides and the formation of [a3-B3] ions, where B3 is the base on the third position, are effective for identification and distinction of pairs of isomeric tetranucleotides. A sufficient number of tetramers and pentamers were studied to enable us to propose a charge-remote mechanism for the formation of site-specific [an-Bn] ion.