Alex Romaschin

Ruptured abdominal aortic aneurysm, a “two-hit” ischemia/reperfusion injury: Evidence from an analysis of oxidative products

PURPOSEnRuptured abdominal aortic aneurysm (RAAA) remains a lethal condition despite improvements in perioperative care. The consequences of RAAA are hypothesized to result from a combination of two ischemia/reperfusion events: hemorrhagic shock and lower torso ischemia. Ischemia/reperfusion results in tissue injury by diverse mechanisms, which include oxygen free radical-mediated injury produced from activated neutrophils, xanthine oxidase, and mitochondria. Oxygen-free radicals attack membrane lipids, resulting in membrane and subsequently cellular dysfunction that contributes to postoperative organ injury/failure. The purpose of this investigation was to quantify the oxidative injury that occurs as a result of the ischemia/reperfusion events in RAAAs and elective AAAs.nnnMETHODSnBlood samples were taken from 22 patients for elective AAA repair and from 14 patients for RAAA repair during the perioperative period. Plasma F(2)-isoprostanes were extracted, purified, and measured with an enzyme immunoassay. Aldehydes and acyloins were purified and quantified. Neutrophil oxidative burst was measured in response to a receptor independent stimulus (phorbol 12-myristate 13-acetate) with luminol-based chemiluminescence.nnnRESULTSnPlasma from patients with RAAAs showed significantly elevated F(2)-isoprostane levels on arrival at hospital and were significantly elevated as compared with the levels of patients for elective repair throughout the perioperative period (two-way analysis of variance, P <.0001). Multiple regression showed a significant relationship between the phagocyte oxidative activity and F(2)-isoprostane levels (P <.013). Total acyloin levels were significantly higher in patients with RAAAs as compared with the levels in elective cases.nnnCONCLUSIONnThe F(2)-isoprostane levels, specific markers of lipid peroxidation, showed that patients with RAAAs had two phases of oxidative injury: before arrival at hospital and after surgery. The significant relationship between the postoperative increases in F(2)-isoprostane levels and the neutrophil oxidant production implicates neutrophils in the oxidative injury that occurs after RAAA. New therapeutic interventions that attenuate neutrophil-mediated oxidant injury during reperfusion may decrease organ failure and ultimately mortality in patients with RAAAs.

Epinephrine increases mortality after brief asphyxial cardiac arrest in an in vivo rat model

Epinephrine may be detrimental in cardiac arrest. In this laboratory study we sought to characterize the effect of epinephrine and concomitant calcium channel blockade on postresuscitation myocardial performance after brief asphyxial cardiac arrest. Anesthesized rats were disconnected from mechanical ventilation, resulting in cardiac arrest. Resuscitation was attempted after 1 min with mechanical ventilation, oxygen, chest compressions, and IV medication. In experimental series 1 and 2, animals were allocated to 10 or 30 &mgr;g/kg epinephrine or 0.9% saline. In series 3, animals received 30 &mgr;g/kg of epinephrine and were randomized to 0.1 mg/kg of verapamil or to 0.9% saline. In series 1 and 3, left ventricular function was assessed using transthoracic echocardiography. In series 2, left atrial pressure was measured. Epinephrine was associated with increased mortality (0/8 [0%] in controls, 4/12 [33.3%] in 10 &mgr;g/kg animals, and 16/22 [72.8%] in 30 &mgr;g/kg animals; P < 0.05), hypertension (P < 0.001), tachycardia (P = 0.004), early transient left atrial hypertension, and dose-related reduction in left ventricular end diastolic diameter (P < 0.05). Verapamil prevented mortality associated with large-dose epinephrine (0% versus 100%) and attenuated early diastolic dysfunction and postresuscitation hypertension (P = 0.001) without systolic dysfunction. Epinephrine appears to be harmful in the setting of brief cardiac arrest after asphyxia.

C5 complement inhibition attenuates shock and acute lung injury in an experimental model of ruptured abdominal aortic aneurysm

Ruptured abdominal aortic aneurysm (RAAA) is associated with a systemic inflammatory response syndrome and multiple organ dysfunction. The potential role of a novel C5 complement inhibitor in attenuation of pathological complement activation and tissue injury was explored in a model of RAAA.

Acute pulmonary injury in a model of ruptured abdominal aortic aneurysm

PURPOSEnThe purpose of this study was to determine whether the combined insults of hemorrhagic shock and aortic clamping simulating ruptured abdominal aortic aneurysm repair had a synergistic effect on the production of pulmonary injury, indicating remote organ injury.nnnMETHODSnAnimals were randomized to one of three groups, infrarenal clamp plus 1 hour of shock, infrarenal clamp plus 2 hours of shock, and supramesenteric clamp plus 1 hour of shock. Each of these groups had four subgroups; sham, shock (mean arterial pressure of 50 mm Hg), clamp, or combined [shock plus clamp]). All animals had a laparotomy with aortic clamping in only the clamp and combined groups. Five hours after clamp removal lung permeability index and neutrophil sequestration were quantified.nnnRESULTSnLung permeability index (6.60 +/- 0.63, p < 0.05 vs all other groups) and neutrophil sequestration (3.72 +/- 0.45 vs sham and clamp, p < 0.05) were significantly increased when shock and supramesenteric clamp were combined. After 1 or 2 hours of shock and infrarenal clamping, no increase in lung permeability index was noted, although neutrophil sequestration was increased in the 2-hour shock group.nnnCONCLUSIONSnThese results demonstrate the additive effect of shock and supramesenteric clamping, which initiated a cascade of injurious events that resulted in a rapid pulmonary injury. The high mortality rate related to remote organ failure in ruptured abdominal aortic aneurysm may be related to the synergy of these two injurious processes.

Rupture of an abdominal aortic aneurysm causes priming of phagocytic oxidative burst

PURPOSEnThe purpose of this investigation was to determine whether rupture and repair of an abdominal aortic aneurysm induced activation of phagocyte oxidant burst, reflecting a systemic inflammatory state, when compared with elective abdominal aortic aneurysm (AAA) repair.nnnMETHODSnBlood samples were harvested from 22 patients with elective AAA and 15 patients with ruptured AAA. Phagocyte oxidant activity was measured in response to a panel of activators with luminol and lucigenin as chemiluminescent substrates. Activity of the complement pathways was measured with plasma levels of C3a des arg.nnnRESULTSnElective AAA repair resulted in significant elevation in phagocyte count and oxidative activity after surgery in response to maximal dose phorbol myristate acetate (PMA) when compared with the baseline sample. In patients with ruptured AAA the oxidative activity of phagocytes was significantly increased in response to both unopsonized zymosan (899.8 +/- 192 ruptured vs 300 +/- 40 elective, p < 0.01) and maximal dose PMA (8769 +/- 2011 vs 3508 +/- 382, p < 0.01) compared with elective cases at the initial sampling. Phagocyte priming has occurred by way of two distinct pathways: receptor-mediated (unopsonized zymosan, CR3 receptor) and receptor-independent (PMA, protein kinase c).nnnCONCLUSIONSnRupture of an AAA resulted in priming of the phagocyte oxidant capacity before operative repair compared with elective AAA. Phagocyte activation is a critical component of the systemic inflammatory response that may contribute to the high incidence of systemic organ dysfunction and death in this patient group.

Interference of IgM paraproteins in the olympus AU800 uric acid assay

INTRODUCTIONnIn the Olympus uric acid procedure, uric acid is converted by uricase to allantoin and hydrogen peroxide, which is reacted in a Trinder reaction to produce a chromophore read bichromatically at 520 and 660 nm. Repeated difficulty was encountered in obtaining uric acid results on samples from myeloma patients with known IgM paraproteins. Large absorbances in sample blanks were due to a visible precipitation observed in the reaction cuvettes.nnnOBJECTIVEnTo alter the Olympus method (OM) to eliminate the interference by IgM, and to verify the modified method (MM).nnnMETHODSnDilution of the sample blank by saline was substituted for water in the MM, with small alterations in the reaction timing sequence necessary to accommodate the instrument requirements.nnnRESULTSnA comparison of uric acid results obtained from nonmyeloma patient samples using the OM and the MM showed a good correlation (r = 0.970), and no statistical difference between the two means using a paired t-test. A similar comparison performed using the samples containing IgA and IgG paraproteins also revealed a good correlation (r = 0.981), and no statistical difference between the two means. Results on IgM containing specimens were assessed indirectly because the samples could not be assayed with the OM. First, removal of detectable levels of proteins using a 20% TCA solution did not affect the measurement of uric acid. Second, protein-free supernatants from IgM containing samples were measured by the OM and compared with the corresponding serum samples measured by the MM. There was good correlation between the two methods (r = 0.945), and no statistical difference between the means using a paired t-test.nnnCONCLUSIONnThe modified method is satisfactory for routine analysis of samples, including those with IgM paraproteins.

A CD18 monoclonal antibody reduces multiple organ injury in a model of ruptured abdominal aortic aneurysm.

The role of CD18 antibody (anti-CD18) in remote and local injury in a model of ruptured abdominal aortic aneurysm repair was investigated. Rats were divided into sham, shock, clamp, and shock + clamp groups. Shock + clamp animals received anti-CD18 or a control monoclonal antibody. One hour of hemorrhagic shock was followed by 45 min of supramesenteric aortic clamping. Intestinal and pulmonary permeability to (125)I-labeled albumin was determined. Myeloperoxidase (MPO) activity, F(2)-isoprostane levels, and transaminases were also measured. Only shock + clamp resulted in statistically significant increases in pulmonary and intestinal permeability, which were associated with significant increases in MPO activity and F(2)-isoprostane levels. Treatment with anti-CD18 significantly decreased intestinal and pulmonary permeability in shock + clamp animals. These reductions were associated with significantly reduced intestinal and hepatic MPO activity and pulmonary F(2)-isoprostane levels and reduced alanine and aspartate aminotransferase levels; however, anti-CD18 had no effect on intestinal or hepatic F(2)-isoprostane levels or on pulmonary MPO activity. These results suggest CD18-dependent and -independent mechanisms of local and remote organ injury in this model of ruptured abdominal aortic aneurysm.The role of CD18 antibody (anti-CD18) in remote and local injury in a model of ruptured abdominal aortic aneurysm repair was investigated. Rats were divided into sham, shock, clamp, and shock + clamp groups. Shock + clamp animals received anti-CD18 or a control monoclonal antibody. One hour of hemorrhagic shock was followed by 45 min of supramesenteric aortic clamping. Intestinal and pulmonary permeability to125I-labeled albumin was determined. Myeloperoxidase (MPO) activity, F2-isoprostane levels, and transaminases were also measured. Only shock + clamp resulted in statistically significant increases in pulmonary and intestinal permeability, which were associated with significant increases in MPO activity and F2-isoprostane levels. Treatment with anti-CD18 significantly decreased intestinal and pulmonary permeability in shock + clamp animals. These reductions were associated with significantly reduced intestinal and hepatic MPO activity and pulmonary F2-isoprostane levels and reduced alanine and aspartate aminotransferase levels; however, anti-CD18 had no effect on intestinal or hepatic F2-isoprostane levels or on pulmonary MPO activity. These results suggest CD18-dependent and -independent mechanisms of local and remote organ injury in this model of ruptured abdominal aortic aneurysm.

Mast cell stabilization improves cardiac contractile function following hemorrhagic shock and resuscitation

Hemorrhagic shock (HS) is associated with cardiac contractile dysfunction. Mast cell (MC) degranulation is hypothesized to mediate the cardiodepressant effect. Cardiac function was assessed after HS and resuscitation (HS/R) with the administration of the MC stabilizers to prevent MC degranulation. Anesthetized male Sprague-Dawley rats were randomized to sham-operated control or HS/R groups and underwent 60 min of HS followed by 2 h of resuscitated reperfusion. Animals in the HS/R groups were randomized to receive cromolyn (5 mg/kg), ketotifen (1 mg/kg), or saline 15 min before shock. Hearts were excised following HS or 2 h of reperfusion, and function was assessed on a Langendorff apparatus. A second group of randomized animals had serial blood samples taken to assess MC degranulation by quantifying levels of serum beta-hexosaminidase. Hearts were excised at 0 min (before HS) and following 60 min of HS (before resuscitation) for a histological evaluation of MC density and degranulation. In vivo MC stabilization using ketotifen and cromolyn improved cardiac peak systolic pressure (P < 0.05), contractility (P < 0.05), and relaxation (P < 0.05) compared with that of HS controls. Serum beta-hexosaminidase increased during HS/R and was inhibited by MC stabilization (P < 0.05). Degranulation was inhibited when assessed by histochemistry and immune fluorescence. The inhibition of MC degranulation can significantly improve cardiac function following HS/R.

An improved assay for plasma methylmalonic acid using chemical ionization gas chromatography mass spectrometry

OBJECTIVESnTo develop a precise and sensitive assay for methylmalonic acid (MMA) using positive chemical ionization gas chromatography mass spectrometry (CI GC-MS), and to illustrate its clinical utility.nnnMETHODSnUsing the developed assay, reference intervals were determined with 108 ambulatory individuals, and potential clinical utility examined in 178 consecutive patients with possible cobalamin deficiency (serum B12<200 nmol/L).nnnRESULTS AND CONCLUSIONSnMethylmalonic acid measured by CI GC-MS was precise (CV: 4-5%), and sensitive (limit of quantitation: 37 nmol/L). In a clinical reference set, 37% of individuals with serum B12 less than 200 pmol/L had plasma MMA concentrations within the reference interval (75-378 nmol/L), rendering cobalamin deficiency unlikely. The observation illustrates that MMA assay may be a useful adjunct test in assessing patients with low serum B12.

Monoclonal Antibody Against CD18 Reduces Intestinal and Pulmonary Injury in a Model of Ruptured Abdominal Aortic Aneurysm

Rupture of an abdominal aortic aneurysm (RAAA) is associated with significant morbidity and mortality due to multiple organ failure after successful repair. Because neutrophils are thought to play a pivotal role in the generation of ischemia-reperfusion injury, the purpose of this study was to determine the role of neutrophils in the generation of local (intestinal) and remote (pulmonary) injury after the combined insults of shock and supramesenteric aortic clamping in a model simulating RAAA repair. Methods Sprague-Dawley rats (450 to 550 g) were hemorrhaged to a mean arterial pressure of 50 mm Hg for 1 hour, followed by 45 minutes of supramesenteric aortic clamping. Animals were given anti-CD18 (4 mg/kg) or control antibody (4 mg/kg) 55 minutes into shock and 5 minutes before clamp placement. Intestinal permeability was determined by measuring I 125 -labeled albumin loss into an isolated, perfused segment of small intestine. Pulmonary permeability (LPI) to albumin-I 125 was determined by bronchoalveolar la-vage. Intestinal and pulmonary myeloperoxidase (MPO) activity and F2-isoprostane levels were measured as indexes of neutrophil sequestration and oxidative stress, respectively. Results In shock plus clamp (S + C) animals there was a significant increase in intestinal plasma protein loss from 7.36 ± 2.14 to 33.66 ± 5.90 mg/g dry intestine ( p p p p p p p p p Conclusions The combination of shock and supramesenteric aortic clamping caused a significant intestinal and pulmonary injury characterized by increased capillary permeability, oxidative stress, and neutrophil sequestration. The administration of anti-CD18 antibody significantly decreased organ injury; however, the results suggest that different neutrophil-mediated mechanisms are responsible for the local intestinal and remote pulmonary injury in this RAAA model.