A Stable Isotope Dilution Nanoflow Liquid Chromatography Tandem Mass Spectrometry Assay for the Simultaneous Detection and Quantification of Glyoxal-Induced DNA Cross-Linked Adducts in Leukocytes from Diabetic Patients.

Glyoxal (gx) is a bifunctional electrophile capable of cross-linking DNA. Although it is present in foods and from the environment, endogenous formation of glyoxal occurs through metabolism of carbohydrates and oxidation of lipids and nucleic acids. Plasma concentrations of glyoxal are elevated in in diabetes mellitus patients compared to nondiabetics. The most abundant 2'-deoxyribonucleoside adducts cross-linked by glyoxal are dG-gx-dC, dG-gx-dA, and dG-gx-dG. These DNA cross-links can be mutagenic by damaging the integrity of the DNA structure. Herein, we developed a highly sensitive and specific assay for the simultaneous detection and quantification of the dG-gx-dC and dG-gx-dA cross-links based on stable isotope dilution (SID) nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selected reaction monitoring mode and using a triple quadrupole mass spectrometer. The entire assay procedure involved addition of the stable isotope standards [15N5]dG-gx-dC and [15N5]dG-gx-dA as internal standards, enzyme hydrolysis to release the cross-links as nucleosides, enrichment by a reversed-phase solid-phase extraction column, and nanoLC-NSI/MS/MS analysis. The detection limit is 0.19 amol for dG-gx-dC and 0.89 amol for dG-gx-dA, which is 400 and 80 times more sensitive, respectively, than capillary LC-NSI/MS/MS assay of these adducts. The lower limit of quantification was 94 and 90 amol for dG-gx-dC and dG-gx-dA, respectively, which is equivalent to 0.056 and 0.065 adducts in 108 normal nucleotides in 50 µg of DNA. In type 2 diabetes mellitus (T2DM) patients (n = 38), the levels of dG-gx-dC and dG-gx-dA in leukocyte DNA were 1.94 ± 1.20 and 2.10 ± 1.77 in 108 normal nucleotides, respectively, which were significantly higher than those in nondiabetics (n = 39: 0.83 ± 0.92 and 1.05 ± 0.99 in 108 normal nucleotides, respectively). Excluding the factor of smoking, an exogenous source of glyoxal, levels of these two cross-linked adducts were found to be significantly higher in nonsmoking T2DM patients than in nonsmoking control subjects. Furthermore, the levels of dG-gx-dC and dG-gx-dA correlated with HbA1c with statistical significance. To our best knowledge, this is the first report of the identification and quantification of glyoxal-derived cross-linked DNA adducts in human leukocyte DNA and their association with T2DM. This SID nanoLC-NSI/MS/MS assay is highly sensitive and specific and it requires only 50 µg of leukocyte DNA isolated from 2-3 mL of blood to accurately quantify these two cross-linked adducts simultaneously. Our assay thus provides a useful biomarker for the evaluation of glyoxal-derived DNA damage.

Stability of glyoxal- and methylglyoxal-modified hemoglobin on dried blood spot cards as analyzed by nanoflow liquid chromatography tandem mass spectrometry.

Blood sampling by the dried blood spot (DBS) technique has become commonly applied in newborn screening. It is often used for analysis of small molecules, such as metabolites. Recently, DBS sampling has been applied for quantification of post-translational protein modifications. Glyoxal and methylglyoxal are two simple oxoaldehydes released from glycated proteins in the Maillard reaction. They are widely distributed in the environment (e.g. cigarette smoke) and found in foods and beverages. Glyoxal and methylglyoxal are shown to react with biomolecules including DNA and proteins. In this laboratory, we previously identified the sites of modification by these two oxoaldehydes in human hemoglobin and found that the extents of modification at certain sites of lysine and arginine residues are significantly higher in type 2 diabetes mellitus patients than in nondiabetic individuals. In this study, we examine the stability of these modifications of hemoglobin stored on DBS cards at room temperature or 4 °C in the ambient air. After hemoglobin was extracted from the DBS cards, it was digested by trypsin and analyzed by nanoflow liquid chromatography coupled with nanospray ionization tandem mass spectrometry. The results show that the extents of all these PTMs are stable within 14 and 21 days when stored on DBS at room temperature and at 4 °C, respectively. Extraction of globin from DBS cards is mostly advantageous for hemolytic blood samples. This assay is sensitive as only a quarter of a DBS card containing ca. 12 µL of blood is required. Thus, it is practically useful to measure the extents of glyoxal- and methylglyoxal-induced hemoglobin modifications from DBS cards.

Guidewire retention following central venous catheterisation: a human factors and safe design investigation.

BACKGROUND: Central Venous Catheterisation (CVC) has occasionally been associated with cases of retained guidewires in patients after surgery. In theory, this is a completely avoidable complication; however, as with any human procedure, operator error leading to guidewires being occasionally retained cannot be fully eliminated. OBJECTIVE: The work described here investigated the issue in an attempt to better understand it both from an operator and a systems perspective, and to ultimately recommend appropriate safe design solutions that reduce guidewire retention errors. METHODS: Nine distinct methods were used: observations of the procedure, a literature review, interviewing CVC end-users, task analysis construction, CVC procedural audits, two human reliability assessments, usability heuristics and a comprehensive solution survey with CVC end-users. RESULTS: The three solutions that operators rated most highly, in terms of both practicality and effectiveness, were: making trainees better aware of the potential guidewire complications and strongly emphasising guidewire removal in CVC training, actively checking that the guidewire is present in the waste tray for disposal, and standardising purchase of central line sets so that differences that may affect chances of guidewire loss is minimised. CONCLUSIONS: Further work to eliminate/engineer out the possibility of guidewires being retained is proposed.