Shades of Blue: A Case Series of Acquired Methemoglobinemia.

Acquired methemoglobinemia (MetHb) is a rare but potentially life-threatening condition that has varied etiology, usually toxin- or drug-induced. We had five cases of acquired methemoglobinemia during six months. Their presentation varied from an asymptomatic state to respiratory distress. The presence of cyanosis and low oxygen saturation (SpO2), despite normal partial pressure of oxygen (PaO2) and chocolate brown-colored blood, were diagnostic clues present in all cases. A high level of methemoglobinemia was detected on arterial blood gas (ABG), confirming the diagnosis. Methylene blue was used as an antidote along with supportive care in symptomatic cases. All these cases of methemoglobinemia recovered completely. A high index of suspicion for methemoglobinemia should be maintained in cases presenting with persistent hypoxia or cyanosis despite normal PaO2.

High-resolution EPR distance measurements on RNA and DNA with the non-covalent Ç´ spin label.

Pulsed electron paramagnetic resonance (EPR) experiments, among them most prominently pulsed electron-electron double resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.

Noncovalent Spin-Labeling of DNA and RNA Triplexes.

Studying nucleic acids often requires labeling. Many labeling approaches require covalent bonds between the nucleic acid and the label, which complicates experimental procedures. Noncovalent labeling avoids the need for highly specific reagents and reaction conditions, and the effort of purifying bioconjugates. Among the least invasive techniques for studying biomacromolecules are NMR and EPR. Here, we report noncovalent labeling of DNA and RNA triplexes with spin labels that are nucleobase derivatives. Spectroscopic signals indicating strong binding were detected in EPR experiments in the cold, and filtration assays showed micromolar dissociation constants for complexes between a guanine-derived label and triplex motifs containing a single-nucleotide gap in the oligopurine strand. The advantages and challenges of noncovalent labeling via this approach that complements techniques relying on covalent links are discussed.

Purine-Derived Nitroxides for Noncovalent Spin-Labeling of Abasic Sites in Duplex Nucleic Acids.

A series of purine-based spin labels was prepared for noncovalent spin-labeling of abasic sites of duplex nucleic acids through hydrogen bonding to an orphan base on the opposing strand and π-stacking interactions with the flanking bases. Both 1,1,3,3-tetramethylisoindolin-2-yloxyl and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were conjugated to either the C2- or C6-position of the purines, yielding nitroxide derivatives of guanine, adenine, or 2,6-diaminopurine. The isoindoline-derived spin labels showed extensive or full binding to abasic sites in RNA duplexes, whereas the TEMPO-derived spin labels showed limited binding. An adenine-derived spin label (5) bound fully at low temperature to abasic sites in both DNA and RNA duplexes when paired with thymine and uracil, respectively, complementing the previously described guanine-derived spin label Ç´, which binds efficiently opposite cytosine. Compound Ç´ was also shown to bind to abasic sites in DNA-RNA hybrids, either in the DNA- or the RNA-strand. Ç´ showed only a minor flanking-sequence effect upon binding to abasic sites in RNA. When the abasic site was placed close to the end of the RNA duplex, the affinity of the spin label Ç´ was reduced; full binding was observed at the fourth position from the duplex end. In summary, spin labels 5 and Ç´ showed full binding to abasic sites in both DNA and RNA duplexes and are promising spin labels for structural studies of nucleic acids by pulsed EPR methods.

Noncovalent and site-directed spin labeling of duplex RNA.

An isoindoline-nitroxide derivative of guanine (Ç´, "G-spin") was shown to bind specifically and effectively to abasic sites in duplex RNAs. Distance measurements on a Ç´-labeled duplex RNA with PELDOR (DEER) showed a strong orientation dependence. Thus, Ç´ is a readily synthesized, orientation-selective spin label for "mix and measure" PELDOR experiments.

Quantitative UPLC-MS/MS assay of urinary 2,8-dihydroxyadenine for diagnosis and management of adenine phosphoribosyltransferase deficiency.

Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary disorder that leads to excessive urinary excretion of 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol or febuxostat reduces DHA production and attenuates the renal manifestations. Assessment of DHA crystalluria by urine microscopy is used for therapeutic monitoring, but lacks sensitivity. We report a high-throughput assay based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for quantification of urinary DHA. The UPLC-MS/MS assay was optimized by a chemometric approach for absolute quantification of DHA, utilizing isotopically labeled DHA as an internal standard. Experimental screening was conducted with D-optimal design and optimization of the DHA response was performed with central composite face design and related to the peak area of DHA using partial least square regression. Acceptable precision and accuracy of the DHA concentration were obtained over a calibration range of 100 to 5000ng/mL on three different days. The intra- and inter-day accuracy and precision coefficients of variation were well within ±15% for quality control samples analyzed in replicates of six at three concentration levels. Absolute quantification of DHA in urine samples from patients with APRT deficiency was achieved wihtin 6.5min. Measurement of DHA in 24h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563ng/mL and 24h excretion of 816, 1327 and 1649mg, respectively. A rapid and robust UPLC-MS/MS assay for absolute quantification of DHA in urine was successfully developed. We believe this method will greatly facilitate diagnosis and management of patients with APRT deficiency.