The use of solid-phase microextraction in conjunction with a benchtop quadrupole mass spectrometer for the analysis of volatile organic compounds in human blood at the low parts-per-trillion level.

The analysis of volatile organic compounds (VOCs) in whole human blood at the low parts-per-trillion level has until recently required the use of a high-resolution mass spectrometer to obtain the specificity and detection limits required for epidemiological studies of VOC exposure in the general public. Because of the expense and expertise required to operate and maintain a high-resolution instrument, the applicability of this method has been limited. These limitations are overcome in a new method using automated headspace solid-phase microextraction (SPME) in conjunction with a gas chromatograph and a benchtop quadrupole mass spectrometer. A combination of SPME and multiple single-ion monitoring minimizes the interferences and chemical noise associated with whole blood samples. This method permits the analysis of 10 VOCs in human blood while simplifying the sample preparation and reducing the possible exposure of the analyst to blood aerosols. Twelve samples can be run successively in a fully automated mode, thus eliminating the need for operator attention. Detection limits are below 50 ppt (pg/mL) for a majority of the VOCs tested with a 5-mL sample.

Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames.

As part of EUROFAN (European Functional Analysis Network), we investigated 21 novel yeast open reading frames (ORFs) by growth and sporulation tests of deletion mutants. Two genes (YNL026w and YNL075w) are essential for mitotic growth and three deletion strains (ynl080c, ynl081c and ynl225c) grew with reduced rates. Two genes (YNL223w and YNL225c) were identified to be required for sporulation. In addition we also performed green fluorescent protein (GFP) tagging for localization studies. GFP labelling indicated the spindle pole body (Ynl225c-GFP) and the nucleus (Ynl075w-GFP) as the sites of action of two proteins. Ynl080c-GFP and Ynl081c-GFP fluorescence was visible in dot-shaped and elongated structures, whereas the Ynl022c-GFP signal was always found as one spot per cell, usually in the vicinity of nuclear DNA. The remaining C-terminal GFP fusions did not produce a clearly identifiable fluorescence signal. For 10 ORFs we constructed 5'-GFP fusions that were expressed from the regulatable GAL1 promoter. In all cases we observed GFP fluorescence upon induction but the localization of the fusion proteins remained difficult to determine. GFP-Ynl020c and GFP-Ynl034w strains grew only poorly on galactose, indicating a toxic effect of the overexpressed fusion proteins. In summary, we obtained a discernible GFP localization pattern in five of 20 strains investigated (25%). A deletion phenotype was observed in seven of 21 (33%) and an overexpression phenotype in two of 10 (20%) cases.

Environmental exposure to volatile organic compounds among workers in Mexico City as assessed by personal monitors and blood concentrations.

Benzene, an important component in gasoline, is a widely distributed environmental contaminant that has been linked to known health effects in animals and humans, including leukemia. In Mexico City, environmental benzene levels, which may be elevated because of the heavy traffic and the poor emission control devices of older vehicles, may pose a health risk to the population. To assess the potential risk, portable passive monitors and blood concentrations were used to survey three different occupational groups in Mexico City. Passive monitors measured the personal exposure of 45 workers to benzene, ethylbenzene, toluene, o-xylene and m-/p-xylene during a work shift. Blood concentrations of the above volatile organic compounds (VOCs), methyl tert-butyl ether, and styrene were measured at the beginning and the end of a work shift. Passive monitors showed significantly higher (p > 0.0001) benzene exposure levels among service station attendants (median = 330 microg/m3; range 130-770) as compared to street vendors (median = 62 microg/m3; range 49-180) and office workers (median = 44 microg/m3, range 32-67). Baseline blood benzene levels (BBLs) for these groups were higher than those reported for similar populations from Western countries (median = 0.63 microg/L, n = 24 for service station attendants; median = 0.30 microg/L, n = 6 for street vendors; and median = 0.17 microgr;g/L, n = 7 for office workers). Nonsmoking office workers who were nonoccupationally exposed to VOCs had BBLs that were more than five times higher than those observed in a nonsmoking U.S. population. BBLs of participants did not increase during the work shift, suggesting that because the participants were chronically exposed to benzene, complex pharmacokinetic mechanisms were involved. Our results highlight the need for more complete studies to assess the potential benefits of setting environmental standards for benzene and other VOCs in Mexico.

Laboratory and Field Assays with Entomopathogenic Nematodes for the Management of Oblique Banded Leafroller Choristoneura rosaceana (Harris) (Tortricidae).

The activity of steinernematid entomopathogenic nematodes against Choristoneura rosaceana was evaluated under laboratory and field conditions. In petri dish trials, all instars were susceptible to Steinernema carpocapsae AII strain with LD(50) values of 13, 5, 3, and 2 infective juveniles for the third-, fourth-, fifth-, and sixth-stage larvae, respectively. Steinernema riobrave 335, S. feltiae UK, S. carpocapsae AII, and S. glaseri 326 caused 85, 55, 45, and 8% mortality of third instars when exposed to the concentration of 25 infective juveniles per dish. When third instars were exposed to S. carpocapsae AII for 0, 1, 4, 8, 12, and 24 hours, larval mortality was 12, 13, 21, 47, 64, and 87%, respectively. At least 8 hours' exposure was required to cause a significant increase in mortality when compared with the control (water) and the 1 and 4-hour exposures. None of the tested adjuvants provided a significant improvement in the average total number and the average number of living S. carpocapsae AII per unit leaf area when compared to the water control. Under field conditions, foliar applications of S. carpocapsae AII at the rate of 2 x 10 infective juveniles/ha provided 37, 19, and 13% larval control. At present, efficacy level and treatment cost preclude nematode applications as a sole treatment against this pest.

Four-color flow cytometric immunophenotypic determination of peripheral blood CD4+ T-lymphocyte counts: a comparison of validity and cost-effectiveness with a two-color method.

The clinical usefulness of monitoring CD4+ T-lymphocyte counts in patients infected with HIV is now well established. The need for accurate, rapid, and cost-effective methods for making these determinations is evident. Recent technologic advances have allowed for the development of a system for the determination of CD4+ T-lymphocyte counts by simultaneous 4-color flow cytometry. A new 4-color 2-tube flow cytometric method for analyzing CD4+ T-lymphocyte subsets in whole blood was compared with a standard 2-color 5-tube method. The new method provides results almost identical to those of the well-established 2-color method used in our clinical laboratory. Statistical analyses indicate very low variability in CD4+ counts between the 2 methods, strongly supporting the usefulness of this new procedure. In addition, the 4-color procedure provides a 15% reduction in the materials cost per test compared with the 2-color method, as well as a marked reduction in the time expenditure of flow cytometry technologists.

Signalling through the leukotriene B4 receptor involves both alphai and alpha16, but not alphaq or alpha11 G-protein subunits.

COS-7 cells transfected with the leukotriene (LT) B4 receptor (BLTR) cDNA were unable to produce LTB4-induced inositol phosphates (IPs) in spite of the presence of endogenous Galphai, Galphaq and Galpha11 proteins. Co-transfection of BLTR with Galpha16, however, resulted in high levels of IP production, which were 17-, 10- and 6-fold higher than with co-transfected Galpha11, Galphaq and Galpha14, respectively. Co-transfection of BLTR with phospholipase C (PLC) beta2, on the other hand, resulted in efficient IP production and co-transfection of BLTR with both Galpha16 and PLCbeta2 resulted in a greater than additive response.

The Second Canadian Consensus Conference on the Management of Patients with Gastroesophageal Reflux Disease.

The Second Canadian Consensus Conference on the Management of Patients with Gastroesophageal Reflux Disease (GERD) was organized by the Canadian Association of Gastroenterology to address major advances in the understanding of the pathophysiology of GERD, to review the new methods of investigation and therapy introduced since the first conference in 1992 and to examine the issue of relevant health economics. The changes that have taken place over the past four years have been sufficiently dramatic to necessitate reassessment of the recommendations made following the first conference. The second conference dealt with the investigation and treatment of uncomplicated GERD and the complex issues of esophageal and extraesophageal complications such as chest pain, Barrett's esophagus, and reflux-related pulmonary and laryngeal disorders. The role of laparoscopic surgery was also discussed. A decision tree for investigation and treatment of patients with GERD was developed. The 38 participants represented a broad spectrum of experience, location of practice and special interests. The distribution of participants conformed to the recommendations of the Canadian Medical Association guidelines for consensus documents in that there should be input from all possible interested parties. A list of the state-of-the-art lectures presented during the conference, the small group sessions, the session chairpersons and participants are appended to this document. CONCLUSIONS. UNCOMPLICATED GERD: GERD with alarm symptoms must be investigated immediately. There was no consensus about when to investigate uncomplicated GERD, ie, whether to perform endoscopy immediately or after initial therapy fails. There was controversy regarding 'step up' (H2 receptor antagonist [H2RA] or prokinetic [PK] first therapy) versus 'step down' therapy (proton pump inhibitor [PPI] first therapy). The majority decision was for short term 'step up' therapy and investigation if symptoms do not improve or recur. Maintenance therapy should be carried out with the initial therapy that was effective. H2RAs and PKs may suffice for maintenance therapy in milder GERD; however, for severe esophagitis, PPIs should be used. SURGERY: Indications for laparoscopic surgery should be the same as for conventional antireflux operations. NONCARDIAC ANGINA-LIKE CHEST PAIN: After exclusion of nonesophageal causes, the majority decided that eight weeks of therapy with a PPI should be performed, while some suggested work-up before a therapeutic test. In the absence of response or recurrence, esophagogastroduodenoscopy (EGD) and, depending on the circumstances, 24 h ambulatory pH/motility may be indicated. BARRETT'S ESOPHAGUS: Only patients who, in case of future discovery of cancer or dysplasia, are able or willing to undergo therapy should have surveillance. In the absence of dysplasia EGD should be performed every two years, and in the presence of mild dysplasia every three to six months. All agreed that for severe dysplasia, esophagectomy or poor risk patients, esophageal mucosal ablation is indicated. ESTRAESOPHAGEAL COMPLICATONS (EECs): Asthma, chronic cough and posterior laryngitis were considered EECs. Although PPIs may decrease symptoms, improvement alone is not diagnostic of the presence of EEC. Ambulatory pH studies with two pH probes or ambulatory pH/motility may be useful in establishing causation. HEALTH ECONOMICS: There are limited data for an economic comparison among the different drugs or between medical and surgical therapy.

Assessment of clinical severity and investigation of uncomplicated gastroesophageal reflux disease and noncardiac angina-like chest pain.

Heartburn, suggesting gastroesophageal reflux, is common. Epidemiological studies have shown that 36% to 44% of adults experience heartburn at least once a month, 14% weekly and 7% once a day. Heartburn and regurgitations are the typical symptoms of gastroesophageal reflux disease (GERD). When present as predominant symptoms, they are quite specific but not very sensitive. Clinical severity of GERD does not predict the severity of the underlying condition. The diagnostic approach to patients with GERD depends on the clinical presentation and the question to be answered -Is abnormal reflux present? Is there mucosal injury? Are symptoms due to reflux? Several techniques such as barium swallow, endoscopy, ambulatory pH monitoring, esophageal manometry and 24 h pH/motility can be used to answer those questions. Barium swallow is not much help in diagnosing reflux esophagitis because reflux can occur in more than 25% of asymptomatic patients. It is most useful in demonstrating structural abnormalities such as strictures and hiatal hernia. The importance of hiatal hernia in the pathogenesis of GERD has been controversial. Recent studies suggest that GERD patients with hiatal hernia present with greater extent of reflux and more severe esophagitis. Endoscopy is the best diagnostic study for mucosal evaluation. Ambulatory 24 h pH monitoring is indicated for patients with atypical symptoms of reflux such as chest pain or pulmonary symptoms, or those who do no respond to therapy. The evaluation of duodenogastroesophageal reflux or alkaline reflux can be measured, but the clinical importance of this test remains controversial. Esophageal manometry allows measurement of the lower esophageal sphincter pressure (LES) and the evaluation of esophageal peristalsis. There is a lack of correlation between LES and reflux esophagitis. The role of peristaltic dysfunction in GERD is unclear, but the high percentage of abnormal contractions suggests a more severe form of GERD. Esophageal motility study can document the presence of effective esophageal peristalsis in patients before antireflux surgery. Twenty-four hour pH/motility is not yet available widely. It is useful in patients who have several daily attacks. There is a correlation with acid reflux in approximately 40% of events. Investigation of noncardiac angina-like chest pain is best achieved by standard esophageal manometry combined with provocative testing. Most laboratories performing these studies use acid perfusion and pharmacostimulation with either bethanechol or edrophonium to reproduce the patient's chest pain during esophageal manometry. Esophageal balloon distension is considered to give the highest yield as a provocative test in patients with angina-like chest pain. It is believed that abnormal esophageal nociception is not simply related to underlying motor dysfunction but also to the presence of a visceral sensory abnormality.

Beam-induced reaction between meta-nitrobenzyl alcohol and dipyridocyanine dyes in liquid-secondary-ion mass spectrometry.

Analyses of cationic dipyridocyanine dyes by liquid-secondary-ion mass spectrometry in a liquid matrix of meta-nitrobenzyl alcohol (rnNBA) provide evidence for beam-induced addition reactions between the sample molecule (C) and the mNBA solvent. The ionic products of these addition reactions formally correspond to [C+mNBA-O2](+), Ic+mNBA-O2-H](+), and [C+mNBA-O2-2H](+). Initial loss of H from the adduct ion extends the conjugation of the adduct into the mNBA ring structure, whereas the final loss of hydrogen is thought to be promulgated by the formation of a benzylic radical stabilized through resonance with the π-electron system of the nitrobenzyl alcohol. Alternatively, two hydrogens may be lost from the alcohol functionality to form an aldehyde.