Use of laboratory testing for prediction of postoperative bleeding volume in cardiovascular surgery.

BACKGROUND: Coagulopathy and following massive bleeding are complications of cardiovascular surgery, particularly occurring after procedures requiring prolonged cardiopulmonary bypass (CPB). Reliable and rapid tests for coagulopathy are desirable for guiding transfusion. Measuring multiple coagulation parameters may prove useful. The purpose of this study is to determine the laboratory parameters predicting massive bleeding. METHODS: In a prospectively collected cohort of 48 patients undergoing cardiovascular surgery, markers of coagulation and fibrinolysis were measured using automated analyzer and their correlations with bleeding volume were determined. RESULTS: Operation time was 318 (107-654) min. CPB time was 181 (58-501) min. Bleeding volume during surgery was 2269 (174-10,607) ml. Number of transfusion units during surgery were packed red blood cells 12 (0-30) units, fresh frozen plasma 12 (0-44) units, platelets 20 (0-60) units and intraoperative autologous blood collection 669 (0-4439) ml. Post-surgery activities of coagulation factors II (FII), FV, FVII, FVIII, FIX, FX, FXI and FXII were decreased. Values of fibrinogen, antithrombin, α2 plasmin inhibitor (α2PI) and FXIII were also decreased. Values of thrombin-antithrombin complex (TAT) were increased. Values of FII, FIX, FXI and α2PI before surgery were negatively correlated with bleeding volume (FII, r = - 0.506: FIX, r = - 0.504: FXI, r = - 0.580; α2PI, r = - 0.418). Level of FIX after surgery was negatively correlated with bleeding volume (r = - 0.445) and level of TAT after surgery was positively correlated with bleeding volume (r = 0.443). CONCLUSIONS: These results suggest that several clinical and routine laboratory parameters of coagulation were individually associated with bleeding volume during cardiovascular surgery. Determining the patterns of coagulopathy may potentially help guide transfusion during cardiovascular surgery.

Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe.

Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10kb) in mtDNA were quantitatively amplified using 0.1ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging.

Development of a mass spectrometric hydroxyl-position determination method for the hydroxyindole metabolites of JWH-018 by GC-MS/MS.

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH-018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography-electron ionization-tandem mass spectrometry (GC-EI-MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole-type SC JWH-081, and speculated that the same approach could be used for the metabolite isomers. Using JWH-018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC-MS/MS. Standard compounds of JWH-018 and its hydroxyindole metabolite positional isomers were first analyzed by GC-EI-MS in full scan mode, which was only able to differentiate the 4-hydroxyindole isomer. Further GC-MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH-018 administered mice and determined the hydroxyl positions to be at the 6-position on the indole ring. GC-EI-MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH-018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.

High-resolution mass spectrometric determination of the synthetic cannabinoids MAM-2201, AM-2201, AM-2232, and their metabolites in postmortem plasma and urine by LC/Q-TOFMS.

High-resolution mass spectrometry and accurate mass measurement by liquid chromatography/quadrupole-time of flight mass spectrometry (LC/Q-TOFMS) was applied to postmortem plasma and urine specimens from an autopsy of a fatal case involving synthetic cannabinoid use, resulting in the detection of three synthetic cannabinoids: MAM-2201, AM-1220, and AM-2232. We searched for their metabolites existing in postmortem plasma or urine by LC/Q-TOFMS and were able to detect N-dealkylated metabolites, defluorinated and further oxidized metabolites of MAM-2201, and some hydroxylated metabolites. Postmortem plasma concentrations of the parent drugs, N-dealkylated metabolites, and fluorinated and further oxidized metabolites of MAM-2201 were measured, and quantitation results revealed site differences between heart and femoral postmortem plasma concentrations of parent drugs and some metabolites, suggesting postmortem redistribution of the synthetic cannabinoids and their metabolites. Quantitation results suggest that defluorination is a major metabolic pathway for MAM-2201, and N-dealkylation is a common but minor pathway for the naphthoylindole-type synthetic cannabinoids in human.

Functional expression of recombinant human macrophage ß-glucan receptor dectin-1 using baculovirus-silkworm expression system.

Human macrophage dectin-1, a type II transmembrane ß-glucan receptor, was expressed as a fusion protein with an N-terminal hexahistidine tag in a baculovirus-silkworm expression system and assayed for binding activity. Recombinant dectin-1 specifically bound to some ß-glucans, and the neck domain and N-linked oligosaccharide chains of human dectin-1 did not affect the ligand binding activity and specificity of the receptor.

Expression of active and inactive recombinant soluble trehalase using baculovirus-silkworm expression system and their glycan structures.

Silkworm soluble trehalase was expressed as a fusion protein with an N-terminal or C-terminal hexahistidine tag in a baculovirus-silkworm expression system and assayed for enzymatic activity. Only N-terminally tagged trehalase showed a high activity. This study is the first to report in vitro functional expression of recombinant insect soluble trehalase.

Interaction of several Ni(III) complexes of peptides with DNA; analysis by DNA-fiber EPR.

EPR spectra of Ni(III) complexes of GGH, GHG, and GHK were obtained by in-situ oxidation of the Ni(II) complexes in DNA-pellet and on DNA-fibers. A species with an apical coordination of nitrogenous base of DNA was detected for Ni(III) GGH. Both GHG and GHK complexes showed the EPR signals of Ni(III) species when the ligand to metal ion ratio was 2:1. The complexes bind as Ni(III)(N6), NI(III)(N5), and Ni(III)(N4) species on DNA.