Evaluation of Combination Drug Therapy for Treatment of Antibiotic-Resistant Inhalation Anthrax in a Murine Model.

Bacillus anthracis is considered a likely agent to be used as a bioweapon, and the use of a strain resistant to the first-line antimicrobial treatments is a concern. We determined treatment efficacies against a ciprofloxacin-resistant strain of B. anthracis (Cipr Ames) in a murine inhalational anthrax model. Ten groups of 46 BALB/c mice were exposed by inhalation to 7 to 35 times the 50% lethal dose (LD50) of B. anthracis Cipr Ames spores. Commencing at 36 h postexposure, groups were administered intraperitoneal doses of sterile water for injections (SWI) and ciprofloxacin alone (control groups), or ciprofloxacin combined with two antimicrobials, including meropenem-linezolid, meropenem-clindamycin, meropenem-rifampin, meropenem-doxycycline, penicillin-linezolid, penicillin-doxycycline, rifampin-linezolid, and rifampin-clindamycin, at appropriate dosing intervals (6 or 12 h) for the respective antibiotics. Ten mice per group were treated for 14 days and observed until day 28. The remaining animals were euthanized every 6 to 12 h, and blood, lungs, and spleens were collected for lethal factor (LF) and/or bacterial load determinations. All combination groups showed significant survival over the SWI and ciprofloxacin controls: meropenem-linezolid (P = 0.004), meropenem-clindamycin (P = 0.005), meropenem-rifampin (P = 0.012), meropenem-doxycycline (P = 0.032), penicillin-doxycycline (P = 0.012), penicillin-linezolid (P = 0.026), rifampin-linezolid (P = 0.001), and rifampin-clindamycin (P = 0.032). In controls, blood, lung, and spleen bacterial counts increased to terminal endpoints. In combination treatment groups, blood and spleen bacterial counts showed low/no colonies after 24-h treatments. The LF fell below the detection limits for all combination groups yet remained elevated in control groups. Combinations with linezolid had the greatest inhibitory effect on mean LF levels.

Retrospective detection of exposure to sulfur mustard: improvements on an assay for liquid chromatography-tandem mass spectrometry analysis of albumin-sulfur mustard adducts.

We here report on the further development of the method comprising the pronase digestion of albumin alkylated by sulfur mustard and the subsequent mass spectrometric analysis of an adducted tripeptide. This includes significant improvements in both the albumin isolation procedure and the automation of the microliquid chromatography-electrospray-tandem mass spectrometric analysis. We also report on the results of a small reference range study, in which we have established that there are no detectable interferences in sera from unexposed individuals.

Measurement of bisphenol A levels in human urine.

We report a new approach for assessing human exposure to bisphenol A (BPA) by measuring BPA in urine after enzymatic deglucuronidation. This method involves addition of (13)C(12)-labeled BPA, enzymatic deconjugation, solid-phase extraction, and derivatization with pentafluorobenzyl bromide. The product of the derivatization is separated by gas chromatography followed by mass spectrometric detection using negative chemical ionization and selected ion monitoring. Using this analysis method, urine samples fortified with both a constant level of labeled BPA and a range of unlabeled BPA levels (0.27-10.6 ng/ml) demonstrated constant percentage recovery. In addition, a range of urine sample volumes (0.25-10.0 ml) with constant amounts of added internal standard produced a linear response (r(2)=0.99). The method limit of detection was 0.12 ng/ml. This method was validated by duplicate analyses using gas chromatography coupled to a high-resolution mass spectrometer.

Levels of methyleugenol in a subset of adults in the general U.S. population as determined by high resolution mass spectrometry.

We developed a sensitive and accurate analytical method for quantifying methyleugenol (ME) in human serum. Our method uses a simple solid-phase extraction followed by a highly specific analysis using isotope dilution gas chromatography-high resolution mass spectrometry. Our method is very accurate; its limit of detection is 3.1 pg/g and its average coefficient of variation is 14% over a 200-pg/g range. We applied this method to measure serum ME concentrations in adults in the general U.S. population. ME was detected in 98% of our samples, with a mean ME concentration of 24 pg/g (range < 3.1-390 pg/g). Lipid adjustment of the data did not alter the distribution. Bivariate and multivariate analyses using selected demographic variables showed only marginal relationships between race/ethnicity and sex/fasting status with serum ME concentrations. Although no demographic variable was a good predictor of ME exposure or dose, our data indicate prevalent exposure of U.S. adults to ME. Detailed pharmacokinetic studies are required to determine the relationship between ME intake and human serum ME concentrations.

Strategies for biological monitoring of exposure for contemporary-use pesticides.

Pesticides are used on a massive scale in the United States. The widespread use of these pesticides has made it virtually impossible for the average person to avoid exposure at some level. Generally, it is believed that low-level exposure to these pesticides does not produce acute toxic effects; however, various cancers and other noncancer health endpoints have been associated with chronic exposure to several groups of pesticides. Therefore, it is imperative that well-designed studies investigate the potential relationship between contemporary pesticide exposure and health effects. For these studies to be accurate, reliable methods for determining individual exposure must be used. Biological monitoring is a useful tool for assessing exposure to some contemporary pesticides. As with any analytical method, biological monitoring entails many difficulties, but, in many instances, they can be overcome by the logical use of available information and information acquired in carefully designed studies. At the Centers for Disease Control and Prevention (CDC), we have acquired extensive experience in the development and application of specific techniques for biological monitoring of a variety of toxicants, including many of the contemporary-use pesticides. We have used these methods to measure the internal dose of pesticides received by people in acute and chronic incidents resulting from both environmental and industrial exposure. Additionally, we have established normative values, or reference ranges, of several pesticides based on measurements of their metabolites in the urine of randomly selected adults in the US population. These data have been successfully used to distinguish overt exposures from 'background' exposure. In this paper, we present several examples of the usefulness of biological monitoring in urine and blood and describe the difficulties involved with developing methods in these matrices. We also present a general strategy, considerations, and recommendations for developing biological monitoring techniques for measuring the internal dose of contemporary-use pesticides.

Current activities at the Centers for Disease Control and Prevention's National Diabetes Laboratory.

In 1997, the Centers for Disease Control and Prevention established the National Diabetes Laboratory in order to help prevent and treat type 1 diabetes. This state-of-the-art laboratory collaborates with research scientists and key national and international organizations throughout the world to identify and study risk factors for type 1 diabetes by developing measurements for glycosylated proteins, developing and evaluating technology for measuring genetic risk factors for the disease, and working to standardize autoantibody measurements. Developing improved technologies for diagnosing and managing diabetes and developing reference materials for properly calibrating and standardizing blood glucose meters are also critical aspects of the laboratory's work. In addition, the laboratory provides quality storage for valuable collections of biologics and other materials and facilitates sharing of specimens, associated epidemiologic data, and test results. Working with our partners in diabetes research, we are improving the diagnosis, treatment, and prevention of type 1 diabetes.

Xanthurenic acid induces gametogenesis in Plasmodium, the malaria parasite.

A small, heat stable chromophore extracted from mosquitoes has recently been implicated as the signal that induces mating of Plasmodium, the malaria parasite. We have used high resolution electrospray mass spectrometry to determine that this gamete activation factor (GAF) has a m/z = 205.0450, suggesting a molecular species composition of C10H7NO4. Xanthurenic acid (XA), a product of tryptophan catabolism, was determined to have an elemental composition, ultraviolet absorbance maxima, and mass spectrum consistent with those characteristics of GAF. XA activated gametogenesis of Plasmodium gallinaceum and P. falciparum in vitro at concentrations lower than 0.5 microM in saline buffered to pH 7.4. A structural analog of XA, kynurenic acid (C10H6NO3), also activated gametogenesis but only at higher concentrations and with less effect. We propose that XA is GAF. This is the first evidence that XA has induction activity.

Epidemic of pediatric deaths from acute renal failure caused by diethylene glycol poisoning. Acute Renal Failure Investigation Team.

CONTEXT: Contaminated pharmaceutical products can result in substantial morbidity and mortality and should be included in the differential diagnosis of deaths of unknown origin. OBJECTIVE: To investigate an outbreak of deaths among children from acute renal failure in Haiti to determine the etiology and institute control measures. DESIGN: Case-control study, cohort study, and laboratory toxicologic evaluation. SETTING: Pediatric population of Haiti. PARTICIPANTS: Cases were defined as Haitian residents younger than 18 years with idiopathic anuria or severe oliguria for 24 hours or longer. Febrile hospitalized children without renal failure were enrolled as control subjects. MAIN OUTCOME MEASURE: The odds of exposure to suspected etiologic agents among cases and controls. RESULTS: We identified 109 cases of acute renal failure among children. The clinical syndrome included renal failure, hepatitis, pancreatitis, central nervous system impairment, coma, and death. Of 87 patients with follow-up information who remained in Haiti for treatment, 85 (98%) died; 3 (27%) of 11 patients transported to the United States for intensive care unit management died before hospital discharge. A locally manufactured acetaminophen syrup was highly associated with disease (odds ratio, 52.7; 95% confidence interval, 15.2-197.2). Diethylene glycol (DEG) was found in patients' bottles in a median concentration of 14.4%. The median estimated toxic dose of DEG was 1.34 mL/kg (range, 0.22-4.42 mL/kg). Glycerin, a raw material imported to Haiti and used in the acetaminophen formulation, was contaminated with 24% DEG. CONCLUSIONS: An epidemic of severe systemic toxicity and deaths from DEG-contaminated acetaminophen syrup occurred in Haiti. Good manufacturing practice regulations should be used by all pharmaceutical manufacturers to prevent such tragedies.

Selectivity of electron-donor- and electron-acceptor-bonded silica packing materials for hydrophobic environmental contaminants in polar and non-polar eluents.

Electron-acceptor-bonded stationary phases, 2-(nitrophenyl)ethylsilyl (NPE) and 3-(p-nitrophenoxy)propylsilyl (NPO), and electron-donor-bonded phases, 3-(N-carbazolyl)propylsilyl (CZP), 2-(1-pyrenyl)ethylsilyl (PYE), and 5-coronenylpentylsilyl (COP), were prepared from silica particles and their selectivities were examined in both polar and non-polar solvents for specific isomers of polychlorodibenzo-p-dioxins (PCDDs), hexachloronaphthalenes (HxCNs) and planar and non-planar polychlorobiphenyl (PCB) congeners. Although no single stationary phase was able to separate all the isomer pairs that are coproduced during the synthesis of the PCDDs and HxCNs, pairs can be separated by selecting a suitable stationary phase and solvent. The separation of mixtures of PCDD isomers were found to be most successful with PYE and NPO phases, which yielded the opposite elution orders for each isomer pair that is produced as a mixture. Similar results were obtained for the HxCN isomers that were separated on PYE and CZP phases. The COP phase provided easier separation of non-ortho-substituted and mono-ortho-substituted PCBs from the other PCBs based on the planarity than PYE phase.

Correlation of environmental carbaryl measurements with serum and urinary 1-naphthol measurements in a farmer applicator and his family.

In exposure or risk assessments, both environmental and biological measurements are often used. Environmental measurements are an excellent means for evaluating regulatory compliance, but the models used to estimate body burden from these measurements are complex. Unless all possible routes of exposure (i.e., inhalation, dermal absorption, ingestion) are evaluated, exposure to a toxicant can be underestimated. To circumvent this problem, measurements of the internal dose of a toxicant in blood, serum, urine, or tissues can be used singularly or in combination with environmental data for exposure assessment. In three separate laboratories, carbaryl or its primary metabolite, 1-naphthol, was measured in personal air, dermal samples, blood serum, and urine from farmer applicators and their families. The usefulness of both environmental and biological data has been demonstrated. For the farmer applicator, the environmental levels of carbaryl would have been sufficient to determine that an exposure had occurred. However, biological measurements were necessary to determine the absorbed dose of each member of the applicator's family. In addition, a correlation between serum and urinary 1-naphthol measurements has been shown; therefore, either matrix can be used to accurately evaluate occupational carbaryl exposure.

Isotope dilution--mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I.

An enzymatic hydrolysis isotope dilution-mass spectrometric method was developed for reference quantification of specific proteins. The analytical procedure involved measuring a reproducibly hydrolyzed peptide (serving as the primary standard) unique to a specific protein. This new mass spectrometric method was evaluated by assessing the concentration of apolipoprotein (apo) A-I in the European Community Bureau of Reference (BCR) lyophilized Certified Reference Material (CRM 393). We used the method to make 96 measurements (4 replicate analyses of 4 enzymatic digests of 6 vials of BCR-CRM 393), which gave an average total protein mass of 1.048 mg (+/- 1.0% at 99% confidence limits). The total overall analytical CV was 3.95%. The results of this evaluation of our model approach to determine the concentration of a specific protein in a purified preparation demonstrated that our new mass spectrometric method can be used to measure apolipoproteins and other specific proteins without the use of epitopic immunoassay methods.

Quantification of polycyclic aromatic hydrocarbons and polychlorinated dibenzo-p-dioxins in human serum by combined micelle-mediated extraction (cloud-point extraction) and HPLC.

Quantification of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated dibenzo-p-dioxins (PCDDs) usually requires preconcentration and cleanup prior to analysis. These procedures often involve using large amounts of toxic organic solvents. The sample preparation from serum is even more complex because of the coextraction of lipids and other nonpolar serum components. We describe the unprecedented use of cloud-point extraction to preconcentrate, extract, and clean up PAHs and PCDDs from human serum using the nonionic surfactant Triton X-100. The samples were analyzed by high-performance liquid chromatography with ultraviolet detection. The phase separation was induced by the addition of salt to the micellar serum solutions. The surfactant-rich phase was treated with acetonitrile and water to precipitate and remove some of the unwanted substances in the serum sample extract without significantly affecting the recoveries of the analytes. The favorable characteristics of cloud-point extraction discussed here strengthen its potential use as an alternative to other techniques of separation.

Possible etiologic agents for toxic oil syndrome: fatty acid esters of 3-(N-phenylamino)-1,2-propanediol.

The etiologic agent(s) that was responsible for the 1981 toxic oil syndrome [TOS] epidemic in Spain has not been identified. Liquid chromatography combined with atmospheric pressure ionization tandem mass spectrometry was used for the analysis of oils associated with TOS. Analyses focused on measuring 3-(N-phenylamino)-1,2-propanediol [PAP], the 3-oleyl ester of PAP [MEPAP], and the 1,2-di-oleyl ester of PAP [DEPAP]. DEPAP and MEPAP were found more frequently and at higher concentrations in TOS case-associated oils than in control oils with odds ratios of 13.7 (95% CI 5.0-38) and 21.9 (95% 6.1-78), respectively. Other fatty acid esters of PAP are also likely to be present in the TOS case-associated oils. More significantly, DEPAP and MEPAP were found in aniline-denatured rapeseed oil refined at ITH, the oil refining company with the clearest link to TOS cases, yet these PAP esters were not detected in unrefined aniline-denatured samples of rapeseed oil delivered to ITH. These results show that the esters of PAP were products of the ITH refining process and were not formed spontaneously during storage. PAP esters were not detected in samples of other aniline-denatured rapeseed oils that were refined elsewhere, and which were not associated with illness. These findings provide strong support for the hypothesis that one or more of the fatty acid esters of PAP were the etiologic agents for TOS.

Accurate optical frequency-interval measurement by use of nonresonant frequency comb generation.

A novel technique for measuring the separation of widely spaced optical frequencies is demonstrated. It relies on frequency comb generation by use of a laser that incorporates a frequency-shifting element. A Nd:YLF laser is used to produce a frequency comb that has a bandwidth of 140 GHz and that contains in excess of 875 discrete frequencies, accurately spaced by 160 MHz. The longitudinal mode spacing of a dual-frequency laser was measured to an accuracy of +/-5 kHz in 3,733,440.0 kHz by use of the technique described here.

FM mode-locked, laser-diode-pumped La(1-x)Nd(x)MgAl(11)O(19) laser.

We report the operation of a laser diode pumped La(1-x)Nd(x)MgAl(11)O(19) laser mode locked by an electro-optic phase modulator. The repetition rate of the laser was 230 MHz, and the average output power was 50 mW, when pumped by a 500-mW broad-stripe laser diode. Transform-limited pulses of 14 ps duration were obtained. We have also demonstrated the FM operation of this laser, with bandwidths of up to 440 GHz being obtained.

Structural classification of carbohydrates in glycoproteins by mass spectrometry and high-performance anion-exchange chromatography.

A general strategy has been developed for determining the structural class (oligomannose, hybrid, complex), branching types (biantennary, triantennary, etc.), and molecular microheterogeneity of N-linked oligosaccharides at specific attachment sites in glycoproteins. This methodology combines mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection to take advantage of their high sensitivity and the capability for analysis of complex mixtures of oligosaccharides. Glycopeptides are identified and isolated by comparative HPLC mapping of proteolytic digests of the protein prior to, and after, enzymatic release of carbohydrates. Oligosaccharides are enzymatically released from each isolated glycopeptide, and the attachment site peptide is identified by fast atom bombardment mass spectrometry (FAB-MS) of the mixture. Part of each reaction mixture is then permethylated and analyzed by FAB-MS to identify the composition and molecular heterogeneity of the carbohydrate moiety. Fragment ions in the FAB mass spectra are useful for detecting specific structural features such as polylactosamine units and bisecting N-acetylhexosamine residues, and for locating inner-core deoxyhexose residues. Methylation analysis of these fractions provides the linkages of monomers. Based on the FAB-MS and methylation analysis data, the structural classes of carbohydrates at each attachment site can be proposed. The remaining portions of released carbohydrates from specific attachment sites are preoperatively fractionated by high-performance anion-exchange chromatography, permethylated, and analyzed by FAB-MS. These analyses yield the charge state and composition of each peak in the chromatographic map, and provide semiquantitative information regarding the relative amounts of each molecular species. Analytically useful data may be obtained with as little as 10 pmol of derivatized carbohydrate, and fmol sensitivity has been achieved. The combined carbohydrate mapping and structural fingerprinting procedures are illustrated for a recombinant form of the CD4 receptor glycoprotein.

Mode locking of a diode-laser-pumped Nd:glass laser by frequency modulation.

We report frequency-modulation mode locking of a diode-laser-pumped Nd:glass laser. We have obtained pulses of 9-psec duration using a lithium niobate phase modulator operating at a repetition rate of 235 MHz. The average output power is 14 mW, for pumping with a 500-mW laser-diode array, and the pulses are approximately 1.4 times transform limited.

Protein and carbohydrate structural analysis of a recombinant soluble CD4 receptor by mass spectrometry.

The primary structure of a soluble form of the CD4 receptor (sCD4) expressed in Chinese hamster ovary cells has been confirmed by mass spectrometric peptide mapping and and tandem mass spectrometry. These studies corroborated 95% of the 369-amino acid-long sequence and established the fidelity of translation of the NH2 and COOH terminal including the absence of "ragged ends." The arrangement of the three disulfide bonds in recombinant sCD4 was also established by mass spectrometry and comparative high performance liquid chromatography mapping and shown to be identical to that expected from previous studies of intrachain disulfide bonding in T4 antigens derived from sheep and mouse. No other arrangements of disulfides were detected. Carbohydrate mapping by mass spectrometry was used to establish that both potential Asn-linked glycosylation sites in sCD4 (Asn271 and Asn300) have oligosaccharides attached. Structural characterization by mass spectrometry and methylation analysis of the heterogeneous family of oligosaccharides at each of the specific attachment sites indicates that the major components of both families of oligosaccharides have the following biantennary structures: (Formula, see text) where m + n = 0-2, and x = 0,1. Minor carbohydrate components having three N-acetylneuraminic acid (NeuAc) groups and an additional hexose-hexosamine unit were detected by high performance anion-exchange chromatography.