Hydrogen sulfide improves resuscitation via non-hibernatory mechanisms in a porcine shock model.

BACKGROUND: Hydrogen sulfide (H2S) has been demonstrated to induce a "suspended animation-like" state in rodent models by reversible inhibition of cellular respiration and marked metabolic suppression and has been proposed as a potential pharmacologic adjunct to resuscitation from shock states. There are few data currently available about the mechanisms and efficacy of H2S in larger animals or humans. We examined H2S as a pharmacologic adjunct to resuscitation in a porcine model of severe traumatic shock. METHODS: Twenty-one adult swine were assigned to three study arms: sham, H2S, and saline vehicle controls (SC). All pigs underwent laparotomy and instrumentation, and the two study arms then underwent a 35% controlled hemorrhage followed by 50 min of truncal ischemia via aortic cross-clamp. H2S (5 mg/kg) or saline was administered immediately before reperfusion, followed by 6 h of resuscitation. Resuscitation requirements, laboratory parameters, end-organ histology, and inflammatory product gene expression (by reverse transcription-polymerase chain reaction) were measured and compared between groups. RESULTS: All animals survived to the 6-h postresuscitation time point. Both treatment arms demonstrated severe shock characterized by fluid and vasopressor requirements, metabolic acidosis, and hypotension compared with sham animals. Animals treated with H2S demonstrated significantly lower resuscitative requirements (total epinephrine 727 versus 3052 µg; P < 0.05), decreased fluid requirements, and lower serum lactate levels (7 versus 10 mmol/L) versus SC. Cardiac output was slightly decreased with H2S treatment but all other hemodynamic and metabolic parameters were equivalent between H2S and C groups. Serum liver and kidney biomarkers were unchanged, but administration of H2S was associated with a significant improvement in histopathologic liver and kidney injury scores compared with SC (both P < 0.05). Both study groups demonstrated significantly increased gene expression of hypoxia-inducible factor 1α and nitric oxide synthase (endogenous nitric oxide synthase, inducible nitric oxide synthase [iNOS]2, iNOS3) relative to sham animals. However, H2S was associated with increased expression of hypoxia-inducible factor 1α and decreased iNOS2 levels compared with SC. CONCLUSIONS: Administration of H2S in a large-animal model of severe traumatic shock resulted in a significant decrease in resuscitative requirements, decreased metabolic acidosis, and less end-organ histologic injury compared with standard resuscitation. H2S did not induce profound metabolic suppression as seen in rodents, and appears to have alternative mechanisms of action in large animals.

Longitudinal analysis of maternal plasma apolipoproteins in pregnancy: a targeted proteomics approach.

Minimally invasive diagnostic tests are needed in obstetrics to identify women at risk for complications during delivery. The apolipoproteins fluctuate in complexity and abundance in maternal plasma during pregnancy and could be incorporated into a blood test to evaluate this risk. The objective of this study was to examine the relative plasma concentrations of apolipoproteins and their biochemically modified subtypes (i.e. proteolytically processed, sialylated, cysteinylated, dimerized) over gestational time using a targeted mass spectrometry approach. Relative abundance of modified and unmodified apolipoproteins A-I, A-II, C-I, C-II, and C-III was determined by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in plasma prospectively collected from 11 gravidas with uncomplicated pregnancies at 4-5 gestational time points per patient. Apolipoproteins were readily identifiable by spectral pattern. Apo C-III(2) and Apo C-III(1) (doubly and singly sialylated Apo C-III subtypes) increased with gestational age (r(2)>0.8). Unmodified Apo A-II, Apo C-I, and Apo C-III(0) showed no correlation (r(2) = 0.01-0.1). Pro-Apo C-II did not increase significantly until third trimester (140 ± 13% of first trimester), but proteolytically cleaved, mature Apo C-II increased in late pregnancy (702 ± 130% of first trimester). Mature Apo C-II represented 6.7 ± 0.9% of total Apo C-II in early gestation and increased to 33 ± 4.5% in third trimester. A label-free, semiquantitative targeted proteomics approach was developed using LTQ-Orbitrap mass spectrometry to confirm the relative quantitative differences observed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in Apo C-III and Apo C-II isoforms between first and third trimesters. Targeted apolipoprotein screening was applied to a cohort of term and preterm patients. Modified Apo A-II isoforms were significantly elevated in plasma from mothers who delivered prematurely relative to term controls (p = 0.02). These results support a role for targeted proteomics profiling approaches in monitoring healthy pregnancies and assessing risk of adverse obstetric outcomes.

Flutamide fails to reduce resuscitation requirements in a porcine ischemia-reperfusion model.

BACKGROUND: Hemorrhagic shock and subsequent resuscitation can lead to ischemia-reperfusion injury, followed by multiorgan failure and death. Flutamide, a vasoactive nonsteroidal antiandrogen compound, is thought to improve tissue and organ perfusion. We tested whether administration of flutamide-cyclodextrin (FLU-CYD) alters physiologic parameters or resuscitation requirements in a porcine model of severe acidosis and shock secondary to combined hemorrhage + ischemia-reperfusion injury. METHODS AND MATERIALS: Fifteen male pigs underwent a 35% blood-volume hemorrhage. Ischemia was induced by cross-clamping the supraceliac aorta for 50 min followed by reperfusion and resuscitation. FLU-CYD complex was administered during aortic clamping. Fluid resuscitation and epinephrine were titrated by protocol to maintain mean arterial pressure ≥40 mm Hg for 6 h. Sequential laboratory results were obtained and serum levels of FLU and 2-hydroxy-flutamide (FLUOH) were measured by mass spectrometry. RESULTS: Mean requirements for injured control swine were 14.6 (± 1.21 standard error of the mean [SEM]) L crystalloid saline and 0.59 (± 0.29 SEM) g epinephrine, compared with 16.30 (± 1.33 SEM) L and 0.54 (± 0.16 SEM) g, respectively, in the FLU-CYD group (both P > 0.05). There were no significant differences in central hemodynamics between control and experimental groups. No significant differences for pH, bicarbonate, fibrinogen, or international normalized ratio were evident. FLU-CYD resuscitation was associated with a significant increase in lactate levels compared with controls (10.1 versus 5.7 mmol/L, P < 0.05). Histologic injury was significantly increased in the livers of FLU-CYD compared with sham (P = 0.022). High serum levels of FLU and the active metabolite FLUOH were measurable throughout the resuscitation period. CONCLUSIONS: Flutamide failed to show any benefit to resuscitation in a model of severe injury and was associated with increased acidosis, hemodilution, and liver injury compared with standard crystalloid resuscitation.

Group B streptococcus serotype prevalence in reproductive-age women at a tertiary care military medical center relative to global serotype distribution.

BACKGROUND: Group B Streptococcus (GBS) serotype (Ia, Ib, II-IX) correlates with pathogen virulence and clinical prognosis. Epidemiological studies of seroprevalence are an important metric for determining the proportion of serotypes in a given population. The purpose of this study was to evaluate the prevalence of individual GBS serotypes at Madigan Healthcare System (Madigan), the largest military tertiary healthcare facility in the Pacific Northwestern United States, and to compare seroprevalences with international locations. METHODS: To determine serotype distribution at Madigan, we obtained GBS isolates from standard-of-care anogenital swabs from 207 women of indeterminate gravidity between ages 18-40 during a five month interval. Serotype was determined using a recently described molecular method of polymerase chain reaction by capsular polysaccharide synthesis (cps) genes associated with pathogen virulence. RESULTS: Serotypes Ia, III, and V were the most prevalent (28%, 27%, and 17%, respectively). A systematic review of global GBS seroprevalence, meta-analysis, and statistical comparison revealed strikingly similar serodistibution at Madigan relative to civilian-sector populations in Canada and the United States. Serotype Ia was the only serotype consistently higher in North American populations relative to other geographic regions (p < 0.005). The number of non-typeable isolates was significantly lower in the study (p < 0.005). CONCLUSION: This study establishes PCR-based serotyping as a viable strategy for GBS epidemiological surveillance. Our results suggest that GBS seroprevalence remains stable in North America over the past two decades.

Molecular weight assessment of proteins in total proteome profiles using 1D-PAGE and LC/MS/MS.

BACKGROUND: The observed molecular weight of a protein on a 1D polyacrylamide gel can provide meaningful insight into its biological function. Differences between a protein's observed molecular weight and that predicted by its full length amino acid sequence can be the result of different types of post-translational events, such as alternative splicing (AS), endoproteolytic processing (EPP), and post-translational modifications (PTMs). The characterization of these events is one of the important goals of total proteome profiling (TPP). LC/MS/MS has emerged as one of the primary tools for TPP, but since this method identifies tryptic fragments of proteins, it has not generally been used for large-scale determination of the molecular weight of intact proteins in complex mixtures. RESULTS: We have developed a set of computational tools for extracting molecular weight information of intact proteins from total proteome profiles in a high throughput manner using 1D-PAGE and LC/MS/MS. We have applied this technology to the proteome profile of a human lymphoblastoid cell line under standard culture conditions. From a total of 1 x 10(7) cells, we identified 821 proteins by at least two tryptic peptides. Additionally, these 821 proteins are well-localized on the 1D-SDS gel. 656 proteins (80%) occur in gel slices in which the observed molecular weight of the protein is consistent with its predicted full-length sequence. A total of 165 proteins (20%) are observed to have molecular weights that differ from their predicted full-length sequence. We explore these molecular-weight differences based on existing protein annotation. CONCLUSION: We demonstrate that the determination of intact protein molecular weight can be achieved in a high-throughput manner using 1D-PAGE and LC/MS/MS. The ability to determine the molecular weight of intact proteins represents a further step in our ability to characterize gene expression at the protein level. The identification of 165 proteins whose observed molecular weight differs from the molecular weight of the predicted full-length sequence provides another entry point into the high-throughput characterization of protein modification.

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics.

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dual-channel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (<20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ion-energy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10(3), and >10(5) for peptide abundances in the overall separation.

Systemic absorption of mitomycin-C when used in refractive surgery.

PURPOSE: To determine whether corneal topical application of mitomycin-C (MMC) results in measurable plasma levels of systemic absorption. SETTING: Madigan Army Medical Center, Refractive Surgery Center, Fort Lewis, Washington, and Micro-Constants Laboratory, San Diego, California, USA. DESIGN: Case-control study. METHODS: The study comprised male and female active-duty soldiers having excimer laser photorefractive keratectomy with MMC. Patients who met inclusion criteria were asked to provide a blood sample immediately after being treated with MMC 0.2 mg/mL (0.02%) for 30 seconds. Human plasma samples were evaluated by liquid chromatography mass spectrometry to determine whether MMC was present. RESULTS: Thirty samples were submitted for evaluation. There was zero detection of MMC in the submitted samples. The quantifiable limit was greater than 10.0 ng/mL. All samples were below this. CONCLUSIONS: In this study of 30 patients with topical application of MMC for refractive surgery, there was no measurable evidence of systemic absorption. Although systemic absorption has been found with use in larger quantities, it was not known whether MMC toxicity concerns could be extrapolated to the refractive surgery population. This information allows counseling of patients on the extremely low likelihood of systemic absorption or toxicity following current techniques for refractive surgery. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry.

Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants, or cancer. Liquid chromatography coupled online with tandem mass spectrometry was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (ranging from 1 to14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of P. aeruginosa and for prokaryotes in general.