Analysis of urine for drugs of abuse using mixed-mode solid-phase extraction and gas chromatography-mass spectrometry.

A method for the simultaneous analysis of urine for the major drugs of abuse is described. The analytical procedure uses solid-phase extraction (SPE), gas chromatography-mass spectrometry (GC-MS) and a semi-automated identification process. It allows simultaneous extraction, derivatization and analysis of acidic, neutral and basic drugs from urine. Urine samples were subjected to enzymatic hydrolysis followed by SPE using Bakerbond narc-2 columns. The eluant was selectively derivatized with N-methyl-bis-trifluoroacetamide (MBTFA) and N-methyl-N-trimethylsilyltrifluoroacetamide + 1% trimethylchlorosilane. Analysis was performed using a GC-MS system operating in full scan mode. A simple macro programme was written to enhance the mass spectra identification capabilities of the MS software by producing extracted ion chromatograms (EIC) for the drugs of interest. Once a suspect compound was indicated by EIC, the mass spectrum of the compound was searched manually against reference libraries for positive identification and the retention time checked against that of the standard. This procedure has increased both the amount and the reliability of information given to clinicians without increasing the cost per sample. The system has been in routine operation for 24 months, processing up to 40 urine samples per day, with a usual turn-around time of 48 h.

Eicosanoid and muscarinic receptor blockade abolishes hyperventilation-induced bronchoconstriction.

This study was designed to test the hypothesis that hyperventilation-induced bronchoconstriction (HIB) results from the combined effects of prostanoid and leukotriene metabolism. A bronchoscope was used in anesthetized dogs to record peripheral airway resistance and HIB before and after combined treatment with inhibitors of cyclooxygenase (indomethacin) and 5-lipoxygenase (MK-0591). Bronchoalveolar lavage fluid (BALF) cells and mediators from hyperventilated and control airways were also measured. Pretreatment with MK-0591 and indomethacin significantly attenuated, but did not abolish, HIB. However, addition of atropine nearly eliminated the residual response. Blockade of eicosanoid metabolism markedly reduced the concentrations of eicosanoids recovered in BALF after hyperventilation. Positive correlations between posthyperventilation BALF prostanoid and epithelial cell concentrations are suggestive of mucosal injury-induced mediator production and release. We conclude that HIB is prevented in the presence of eicosanoid and muscarinic-receptor blockade and that both classes of eicosanoids contribute similarly to the development of HIB.

Intended and stray radiofrequency electrical currents during resectoscopic surgery.

STUDY OBJECTIVE: To test the hypothesis that electrical burns of the genital tract and urethral strictures after hysteroscopic endometrial ablation and transurethral prostatectomy, respectively, are related to capacitive coupling and/or stray currents induced by intact and defective electrodes and/or resectoscopes. DESIGN: Basic in vitro measurements. SETTING: Laboratory. MATERIALS: Porcine muscle and liver, resectoscope, electrosurgical unit (ESU), and ESU analyzer. INTERVENTION: We measured electrical coagulation and cutting currents of rollerball and loop electrodes and the external sheath of the resectoscope from 80 to 200 W through a resistance load of 200 and 250 ohms, using intact electrodes and conditions simulating potential insulation defects along the shaft of the electrodes. MEASUREMENTS AND MAIN RESULTS: Approximately 20% to 25% of current was induced by capacitive coupling to the resectoscope sheath. Touching porcine muscle or liver with small areas of the sheath while the generator was activated resulted in superficial tissue burn. Surrounding large segments of the sheath with tissue did not result in visible burns, indicating that under normal conditions the sheath acts as a dispersive electrode. Defective insulation of distal segments of the electrodes resulted in 100% transfer of current to the sheath and caused extensive electrical burns of tissue in contact with the sheath. CONCLUSIONS: Capacitive coupled currents induced by intact resectoscopes and electrodes may cause thermal injury to surrounding tissue during prolonged resectoscopic surgery. Stray currents from defective insulation of the electrodes result in direct coupling of current to the telescope and sheath and cause extensive burns of surrounding tissues in contact with the sheath.

Genital tract electrical burns during hysteroscopic endometrial ablation: report of 13 cases in the United States and Canada.

We investigated 13 alleged thermal injuries to the genital tract of women undergoing hysteroscopic endometrial ablation. Possible mechanisms proposed to explain these injuries are hot-weighted speculum, povidone-iodine scrub solution, inadequate rinsing of Cidex sterilizing solution, and electrical burns. The history, nature, and distribution, as well as experimental evidence strongly support the hypothesis that these injuries are electrical due to capacitive coupled currents induced onto the sheath of the resectoscope, and/or stray currents generated by arcing or direct coupling from defective electrode insulation to the telescope, electrifying the entire resectoscope.

Mucosal injury and eicosanoid kinetics during hyperventilation-induced bronchoconstriction.

Bronchoalveolar lavage (BAL) of canine peripheral airways was performed at various times after hyperventilation, and BAL fluid (BALF) cell and mediator data were used to evaluate two hypotheses: 1) hyperventilation-induced mucosal injury stimulates mediator production, and 2) mucosal damage is correlated with the magnitude of hyperventilation-induced bronchoconstriction. We found that epithelial cells increased in BALF immediately after a 2- and a 5-min dry air challenge (DAC). Prostaglandins D(2) and F(2alpha) and thromboxane B(2) were unchanged immediately after a 2-min DAC but were significantly increased after a 5-min DAC. Leukotriene C(4), D(4), and E(4) did not increase until 5 min after DAC. Hyperventilation with warm moist air did not alter BALF cells or mediators and caused less airway obstruction that occurred earlier than DAC. BALF epithelial cells were correlated with mediator release, and mediator release and epithelial cells were correlated with hyperventilation-induced bronchoconstriction. These observations are consistent with the hypothesis that hyperventilation-induced mucosal damage initiates peripheral airway constriction via the release of biochemical mediators.

Venous allografts prepared from stripped long saphenous vein. Is there a need for antibiotic sterilisation?

AIM: Can useful lengths of vein be retrieved from varicose vein stripping procedures; is it necessary to sterilise this tissue prior to use as vein allografts? METHOD: Stripped long saphenous vein was retrieved at operation. Vein samples were cultured using direct plate inoculation and enrichment culture. Further samples were immersed in two low concentration antibiotic solutions and recultured. Smooth muscle viability was assessed after antibiotic immersion and storage by cryopreservation. RESULTS: High quality vein could be retrieved by vein stripping. Vein segments grew skin commensals on enrichment culture despite negative cultures with standard media plate inoculation (Chi-squared = 53.34 1 d.f. p < 0.001). Low concentration antibiotic solutions sterilised processed vein. Smooth muscle cell viability was reduced by cryopreservation, Mann-Whitney p = 0.008 (control 98% S.E. 0.93 vs. cryopreserved 64% S.E. 6.58), but prior exposure to antibiotics did not compound this effect. CONCLUSION: Useful lengths of vein grafts can be retrieved from varicose vein stripping procedures. Venous segments are frequently contaminated by skin commensals. Enrichment culture is required to detect contamination. Low concentration antibiotics sterilise venous-tissue without affecting smooth muscle cell viability.

ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.