Potential dysregulation of the pyruvate dehydrogenase complex by bacterial toxins and insulin.

BACKGROUND: The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl CoA, effectively controlling the entrance of glycolysis products into aerobic metabolism. Because hyperlactatemia is one of the hallmarks of sepsis, we hyphothesized that gram-positive and negative bacterial toxin treatment will interfere with mRNA levels of regulatory enzymes of the PDC and overall enzyme activity in hepatocytes. METHODS: HEP G2 hepatocarcinoma cells were incubated for 24 hours in the presence of lipopolysaccaride (LPS) or lipoteichoic acid. Total RNA was then isolated and message RNA levels for both pyruvate dehydrogense kinase 4 and phosphatase 2 were determined by RTPCR. Amplified DNA fragments were visualized by ethidium bromide in agarose gels and densitometry of the bands was performed. Data were then normalized to the housekeeping gene, GAPDH. Enzyme activity was then determined by capturing intact PDC on nitrocellulose membranes then determining PDC-dependent production of NADH. RESULTS: LPS treatment led to a time dependent increase in PDK4 message while decreasing PDP2 levels. Enzyme activity, in these cells, also significantly decreased 24 hours after exposure to LPS. Cells cultured in the presence of lipoteichoic acid and insulin exhibited differing message ratios and activity levels when evaluated at 4 hours, but at 24 hours shifted to mimic those observed in LPS treated cells. CONCLUSION: This data may indicate that exposure to bacterial cell wall components and insulin could create cellular environments that result in a build-up of lactate.

The effect of storage on the accumulation of oxidative biomarkers in donated packed red blood cells.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a life-threatening condition characterized by oxidative stress. Longer storage times of packed red blood cells (PRBC) and other blood products have been implicated with an increased risk in developing TRALI in transfused patients. METHODS: A total of 10 units of blood containing PRBC stored in citrate-phosphate-dextrose buffer at 4 degrees C were included in the study. At Bonfils Blood Center (Denver, CO), samples were collected on storage day 1 and day 42. Samples were immediately centrifuged, and the supernatants were collected and stored at -80 degrees C until further analysis. Oxidation-reduction potential and protein oxidation were measured in both the day 1 and day 42 samples. RESULTS: Oxidation-reduction potential significantly increased (p < 0.05) in the day 42 sample (98.1 mV +/- 21.9 SD) versus the day 1 sample (62.6 mV +/- 21.5 SD). The oxidation of human serum albumin increased by 63.6% during the storage time. Other serum proteins such as apolipoprotein A1 and transthyretin demonstrated similar increases in oxidation. Also, proteins with a cleaved C-terminal amino acid were observed indicating the presence of carboxypeptidase activity, a marker of inflammation. CONCLUSIONS: The presence of an oxidative environment in transfused PRBC increases with storage time. This could partially explain the increased risk of developing TRALI related to the transfusion of older blood products.

Mechanisms of delayed wound healing by commonly used antiseptics.

BACKGROUND: The cytotoxic effects of antiseptics on pivotal cell types of the healing process have been well documented. The purpose of our investigation was to explore the ability of subcytotoxic levels of antiseptics to interfere with fibroblast function. METHODS: Cell proliferation assays were performed by culturing fibroblasts in the presence of commonly used antiseptics. Migration was evaluated using scratch assays in which monolayers were "wounded" and cellular movement was monitored by digital photography. Matrix metalloproteinase (MMP) release was analyzed by zymography. RESULTS: H2O2 and povidone-iodine reduced both migration and proliferation of fibroblasts in a dose-dependent fashion. Treatment with silver-containing antiseptics and chlorhexidine exhibited reductions in proliferation at high concentrations, but enhanced growth at lower doses. Silver-containing compounds and chlorhexidine also proved to be the least detrimental to migration in these assays. metalloproteinase release from the cells was differently affected depending on the dosage and class of antiseptic applied. CONCLUSIONS: When debridement of the wound bed is not sufficient to reduce bacterial loads, the application of broad-spectrum antiseptics maybe indicated. Our data would suggest that H2O2 and iodine are poor choices, potentially retarding the contribution of fibroblasts to the healing process. Silver sulfadiazine and chlorhexidine, at levels still proven to be bactericidal, had fewer detrimental effects on fibroblast activity in these assays. The silver-containing antiseptics may even increase the proliferative potential of these cells in culture.

Clinical utility of the cobalt-albumin binding assay in the diagnosis of intestinal ischemia.

Currently, the rapid diagnosis of mesenteric ischemia is problematic because of the nonspecificity of most laboratory assays and the unreliability of physical examinations. The evaluation of the cobalt-albumin binding assay (CABA) as a diagnostic marker for short-term risk stratification of emergency department patients presenting with symptoms of intestinal ischemia is reported. This preliminary study includes patients scheduled for exploratory laparotomy with symptoms of ischemic bowel and/or bowel obstruction. Approximately 10 mL of blood was drawn from each patient 1 hour preoperatively into a serum separator gel tube. After 30 minutes of clotting time, serum was collected and frozen at -80 degrees C. The CABA test was performed on the samples by an investigator blinded to the patient's condition, and values were compared with the clinical and pathologic diagnosis of ischemic bowel postoperatively. CABA test values are reported as absorbance units (ABSU) at 470 nm. Of the 26 patients enrolled in the study, 12 were clinically diagnosed with intestinal ischemia. These patients had significantly higher CABA test values (0.52 ABSU +/- 0.04 SEM) than patients without intestinal ischemia (0.31 ABSU +/- 0.02 SEM, p = 0.00023). Only two false-positives and no false-negatives were recorded. This resulted in a sensitivity of 100% and a specificity of 85.7% for the CABA test for these particular samples. The CABA test could be a useful tool for clinicians in the risk stratification of intestinal ischemia.

Mass spectrometry analysis of urine and catheter of a patient with purple urinary bag syndrome.

INTRODUCTION: Purple urinary bag syndrome (PUBS) is considered to be a benign condition observed in the urinary catheter and bag in some catheterized patients with urinary tract infections. This syndrome is usually reported to occur in alkaline urine. CASE REPORT: We report of a catheterized patient with PUBS and slightly acidic urine (pH 6-6.5). A novel analysis method was developed using high pressure liquid chromatography and mass spectrometry (HPLC/MS) to detect compounds that are thought to be associated with PUBS. Urine, urinary sediment, and the plastic collection system were assayed and quantitated using these methods. The potential toxicity of one of these compounds, indoxyl sulfate, is discussed. CONCLUSIONS: The presence of PUBS in a catheterized patient with slightly acidic urine is reported. A novel method for the analysis of chemical components of PUBS and the first direct confirmation of the presence of indigo in the urine sediment and collecting system are described.

The formation and rapid clearance of a truncated albumin species in a critically ill patient.

INTRODUCTION: Hypoalbuminemia is known to occur in critically ill patients and is associated with increased mortality. We observed a potentially novel, partial explanation for the hypoalbuminemia noticed in a severely traumatized patient. CASE REPORT: We report of a severely, multi-system traumatized patient in whom hypoalbuminemia was present (1-2 g/dl). The plasma albumin (HSA) was analyzed by liquid chromatography/positive electrospray ionization mass spectrometry. A high percentage of a truncated albumin that lost its carboxy terminal amino acid leucine (HSA-L) associated with a 10-fold increase in plasma carboxypeptidase A (CPA) activity (R(2)=0.994) were found. We estimated the half life of this truncated albumin species to be <80 h. CONCLUSIONS: The increased CPA activity encountered following a traumatic event and subsequent rapid clearance of the resulting HSA-L from plasma might be a contributing factor to the hypoalbuminemia observed in the critically ill patients.

Severe systemic immune response syndrome, low plasma paraoxonase activity, and a new albumin species in a traumatized patient with Gaucher's disease.

INTRODUCTION: Gaucher's disease (GD) is an inborn error, autosomal recessive lysosomal lipid storage disorder characterized by the lack of the enzyme glucocerebrosidase. We observed some abnormalities in the plasma of a traumatized patient with GD. CASE REPORT: We report of a traumatized patient with GD that developed a severe systemic immune response during the course of an extended hospital stay. Plasma paraoxonase (PON) activity was assayed and found to be extremely low possibly due to the existence of GD in this particular patient. Also, a potentially novel post-translational modification (PTM) of albumin was noticed in the patient's plasma that coincided with enzyme replacement therapy (ERT) with Cerezyme. CONCLUSIONS: The decreased plasma PON activity measured might be a contributive factor in the development of an accentuated systemic immune response in a traumatized patient with GD. A modified albumin species could serve as a biomarker for ERT in Gaucher patients.

The association between Chance fractures and intra-abdominal injuries revisited: a multicenter review.

The association between Chance fractures and intra-abdominal injuries is reported to be as high as 89 per cent. Because prior studies were small series or case reports, we conducted a multicenter review to learn the true association between Chance fractures and intra-abdominal injuries as well as diagnostic trends. Trauma registry data, medical records, and radiology reports from 7 trauma centers were used to characterize 79 trauma patients with Chance fractures. Initial methods of abdominal assessment were computed tomography (CT) scan (79%), clinical examination (16%), and diagnostic peritoneal lavage (DPL) (5%). Twenty-six (33%) patients had intraabdominal injuries of which hollow viscus injuries predominated (22%). Twenty patients (25%) underwent laparotomy. The presence of an abdominal wall contusion and automobile restraint use were highly predictive of intra-abdominal injury and the need for laparotomy. The association between a Chance fracture and intra-abdominal injury is not as high as previously reported. CT scan has become the primary modality to assess the abdominal cavity of patients with Chance fractures, whereas the role of DPL has diminished.

Lipid peroxidation and the thiobarbituric acid assay: standardization of the assay when using saturated and unsaturated fatty acids.

Saturated fatty acids are less vulnerable to lipid peroxidation than their unsaturated counterparts. In this investigation, individual fatty acids of the C(16), C(18) and (20) families were subjected to the thiobarbituric (TBA) assay. These fatty acids were chosen based on their degree of saturation and configuration of double bonds. Interestingly, an assay threshold was reached where increasing the fatty acid concentration resulted in no additional decrease in the TBARS concentrations. Therefore, the linear range of TBARS inhibition was determined for fatty acids in the C(16) and C(20) families. The rate of TBARS inhibition was greater for the saturated than for unsaturated fatty acids, as measured from the slope of the linear range. These findings demonstrate the need to standardize the TBARS assay using multiple fatty acid concentrations when using this assay for measuring in vitro lipid peroxidation.

The Fort Hood Massacre: Lessons learned from a high profile mass casualty.

BACKGROUND: On November 5, 2009, an army psychiatrist at Fort Hood in Killeen, TX, allegedly opened fire at the largest US military base in the world, killing 13 and wounding 32. METHODS: Data from debriefing sessions, news media, and area hospitals were reviewed. RESULTS: Ten patients were initially transferred to the regional Level I trauma center. The remainder of the shooting victims were triaged to two other local regional hospitals. National news networks broadcasted the Level I trauma center's referral phone line which resulted in more than 1,300 calls. The resulting difficulties in communication led to the transfer of two victims (one critical) to a regional hospital without a trauma designation. CONCLUSIONS: Triage at the scene was compromised by a lack of a secure environment, leading to undertriage of several patients. Overload of routine communication pathways compounded the problem, suggesting redundancy is crucial. LEVEL OF EVIDENCE: Prognostic study, level V.

Phthalate esters used as plasticizers in packed red blood cell storage bags may lead to progressive toxin exposure and the release of pro-inflammatory cytokines.

Phthalate esters (PE's) are plasticizers used to soften PVC-based medical devices. PE's are the most abundant man-made pollutants and increase the risk of developing an allergic respiratory disease or a malignancy. The leaching of PE's in donated packed red blood cells (PRBC) during storage was assessed. PRBC transfusion bags containing CPD/AS-1 (ADSOL) buffer were analyzed. Samples were collected on storage day 1 and day 42. Two PE's, di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP), were measured by liquid chromatography coupled to mass spectrometry (LCMS). Interleukin-8 (IL-8) was measured by standard ELISA techniques. DEHP significantly increased from 34.3 microM (+/-20.0 SD) on day 1 to 433.2 microM (+/-131.2 SD) on day 42, a 12.6-fold increase. Similarly, MEHP significantly increased from 3.7 microM (+/-2.8 SD) on day 1 to 74.0 microM (+/-19.1 SD) on day 42, a 20.2-fold increase. Also, DEHP and MEHP increased the release of IL-8 from human umbilical vein endothelial cells (HUVEC). The transfusion of older units of PRBC could lead to an accumulation of PE's possibly resulting in inflammation and other effects. This accumulation could be exacerbated due to the decreased metabolism of PE's since trauma patients have a lower esterase activity, the enzymes responsible for metabolizing PE's. The effect of oxidative stress caused by PE's is discussed as a potential mechanism for increases in inflammation caused by older units of PRBC.

Heterogeneity and oxidation status of commercial human albumin preparations in clinical use.

OBJECTIVE: Human serum albumin is indicated for the treatment of shock, acute restoration of blood volume, and in hypoalbuminemia. Conflicting reports are found in the literature for the clinical safety and efficacy of human serum albumin administration to critically ill patients. We sought to analyze various commercially available albumin preparations for common, posttranslational modifications. DESIGN: Analysis of six commercially available albumin preparations for clinical use. SETTING: Trauma research laboratory. SUBJECTS: Commercially available human serum albumin preparations and healthy volunteers. INTERVENTIONS: Six commercially available human serum albumin preparations were analyzed by high-performance liquid chromatography. The presence of various posttranslational modifications was identified by positive electrospray ionization, time-of-flight mass spectrometry. Three different lots from three preparations were also analyzed to assess variability within lots from the same manufacturer. Also, for the purpose of comparison, human serum albumin was analyzed in the plasma of healthy volunteers. MEASUREMENTS AND MAIN RESULTS: The six human serum albumin preparations analyzed contained a high percentage (57.2 +/- 3.3%) of bound Cys34 (oxidation of cysteine in position 34 on the human serum albumin molecule) in comparison to the plasma human serum albumin from healthy volunteers (22.9 +/- 4.8%). Lot-to-lot variability in native human serum albumin ranged between 4.8% and 11.2% in three separate commercial albumins. Significant differences existed among the various commercial preparations in other posttranslational modifications of albumin. CONCLUSIONS: Human serum albumin species with a bound Cys34 account for a large percentage of the composition of human serum albumin preparations used for the treatment of critically ill patients. Also, the variability within lots from the same manufacturer is significant. Consequences of the administration of these oxidized forms of human serum albumin to critically ill patients warrants further investigation.

An anti-inflammatory role for N-acetyl aspartate in stimulated human astroglial cells.

Although N-acetyl-L-aspartate (NAA) has been shown to be important to myelin synthesis and osmotic regulation, the biological rationale for the high levels of NAA found in the brain remains unknown. Here, a human astroglial cell line (STTG) was treated with NAA and stimulated with ionomycin, ionomycin/PMA, or IL-1beta. PGE(2) levels in ionomycin-stimulated STTG cells decreased by 76% and > 95% at NAA concentrations of 10 and 20mM, respectively. NAA also decreased the levels of COX-2 protein and activated NF-kappaB in IL-1beta-stimulated STTG cells but had little effect on unstimulated cells. Also, NAA significantly decreased intracellular calcium levels in ionomycin/PMA-stimulated cells. NAA had no effect on total COX-2 activity or COX-2 mRNA. Acetylation of IkappaBalpha kinase, an acetylation target of aspirin, was not observed when NAA was present. These results demonstrate that NAA appears to be important in the modulation of inflammation in the human STTG astroglial cell line. The results of these findings are discussed in relation to neuronal pathologies that exhibit abnormal NAA levels within the brain.