Are compatible solutes compatible with biological treatment of saline wastewater? Batch and continuous studies using submerged anaerobic membrane bioreactors (SAMBRs).

This study investigated fundamental mechanisms that anaerobic biomass employ to cope with salinity, and applied these findings to a continuous SAMBR. When anaerobic biomass was exposed to 20 and 40 g NaCl/L for 96 h, the main solute generated de novo by biomass was trehalose. When we separately introduced trehalose, N-acetyl-ß-lysine and potassium into a batch culture a slight decrease in sodium inhibition was observed. In contrast, the addition of 0.1 mM and 1 mM of glycine betaine dramatically improved the adaptation of anaerobic biomass to 35 g NaCl/L, and it continued to enhance the adaptation of biomass to the salt for the next three batch feedings without further addition. No shift in archaeal microbial diversity was found when anaerobic biomass was exposed in batch mode to 35 g NaCl/L for 360 h, and no changes were found when glycine betaine was added. The dominant species identified under these conditions were Methanosarcina mazeii and Methanosaeta sp. The addition of 5 mM glycine betaine to a continuous SAMBR at 12 h hydraulic retention time (HRT), and operation in batch mode for 2 days can significantly enhance saline (35 g NaCl/L) synthetic sewage degradation. In addition, the injection of 1 mM of glycine betaine into a SAMBR for five subsequent days also significantly enhanced dissolved organic carbon (DOC) removal from sewage under these conditions. The main compatible solutes generated by anaerobic biomass after 44 days exposure to 35 g NaCl/L in a SAMBR were N-acetyl-ß-lysine and glycine betaine. Finally, the addition of 1 mM glycine betaine to the medium was beneficial for anaerobic biomass in batch mode at 20 °C under saline and non saline conditions.

High-dose tirofiban with enoxaparin and inflammatory markers in high-risk percutaneous intervention.

AIM: The study assessed the benefit of high bolus dose tirofiban (HD-tirofiban) with enoxaparin compared with HD-tirofiban with unfractionated heparin (UFH). The study examined markers of platelet activation, thrombin generation and inflammation. MATERIALS AND METHODS: The study is a prospective single centre open-label trial of patients with high-risk acute coronary syndrome treated with percutaneous intervention (PCI) who were randomized to anticoagulation with UFH or enoxaparin with HD-tirofiban (25 microg kg(-1) bolus). This study measured a panel of platelet activation markers, inflammatory biomarkers and thrombus generation between the two groups. RESULT: Sixty patients undergoing high-risk PCI were enroled in the study. Platelet inhibition as assessed by whole blood aggregometry following HD-tirofiban infusion was similar in both the UFH and enoxaparin groups. CD40 ligand expression on platelets was significantly reduced following PCI with HD-tirofiban and either UFH or enoxaparin. Following PCI, there were significant reductions measured in other markers of platelet activation including PAC-1, P selectin, factor V/Va, platelet-monocyte aggregates and monocyte expression of Mac-1 as determined by analysis of venous blood samples using flow cytometry. Prothrombin fragment 1+2, D-dimer, von Willebrand factor and high sensitive C-reactive protein levels were significantly less post PCI in the enoxaparin group compared with those patients receiving UFH. CONCLUSION: The combination of HD tirofiban with enoxaparin resulted in an attenuated inflammatory response when compared with that of the combination of HD tirofiban with UFH.

Platelet-monocyte aggregates predict troponin rise after percutaneous coronary intervention and are inhibited by Abciximab.

BACKGROUND: Platelet-monocyte aggregates and other markers of platelet activation were investigated before and after percutaneous coronary intervention (PCI) with abciximab therapy. The study sought to assess the relationship between the level of platelet-monocyte aggregation and increases in cardiac troponin I post coronary intervention. METHODS: Blood samples were collected from 40 patients before PCI and 10 min after abciximab administration. These were tested for platelet activation markers by flow cytometry. Cardiac troponin I levels were assayed at baseline and at 24 h post PCI. RESULTS: Compared to healthy controls, patients with coronary artery disease had elevated markers of platelet activation including platelet-monocyte aggregates, P-selectin and PAC-1 (a marker specific for activated glycoprotein IIb/IIIa) prior to PCI. Increased levels of platelet-monocyte aggregates before PCI were associated with increased expression of P-selectin on the platelet surface. Abciximab therapy reduced platelet-monocyte aggregate levels but had no effect on P-selectin expression. The high levels of expression of activated glycoprotein IIb/IIIa (PAC-1) on platelets prior to PCI was reduced with abciximab therapy. Patients with higher levels of platelet-monocyte aggregates prior to PCI were more likely to develop an elevation of cardiac troponin I during the 24 h after PCI. CONCLUSIONS: Increased levels of platelet-monocyte aggregates may predict patients at risk for troponin elevation following PCI and identify those most likely to benefit from abciximab.

Heparin monitoring during cardiac surgery. Part 1: Validation of whole-blood heparin concentration and activated clotting time.

There is limited published data on the agreement between techniques for monitoring heparin levels. The aim of this study was to validate the Hepcon/HMS, with particular focus on the agreement with laboratory anti-Xa assay. The performances of two ACT instruments--Hemochron and HemoTec--were also evaluated, including an assessment for interchangeability. Blood samples from 42 adult cardiopulmonary bypass (CPB) patients were analysed for activated clotting time (ACT), whole-blood heparin concentration (Hepcon/HMS) and anti-factor Xa (anti-Xa) plasma heparin concentration. Agreement between measures was determined using the method of Bland and Altman. Simple analysis of agreement between the Hepcon and anti-Xa heparin revealed the Hepcon has a mean bias of -0.46 U/mL, with the limits of agreement +/- 1.12 U/mL. The comparison between ACT instruments indicated a mean difference of -96 seconds for the HemoTec, with limits of +/- 265 seconds. The Hepcon/ HMS instrument displayed satisfactory agreement with anti-Xa plasma heparin concentration, as the expected variation would not be expected to cause problems in the clinical setting. Agreement between the two measurements of ACT may be satisfactory, provided each is assigned a different target value.

Heparin monitoring during cardiac surgery. Part 2: Calculating the overestimation of heparin by the activated clotting time.

Activated clotting time (ACT) values were converted to heparin concentration, enabling an assessment of the accuracy of the ACT and a quantification of the prolongation imposed by bypass. Blood samples were obtained from 42 adult cardiopulmonary bypass (CPB) patients before and during bypass surgery. Samples were analysed for ACT (HemoTec ACT) and anti-factor Xa (anti-Xa) plasma heparin concentration. The mean heparin concentration calculated before bypass was an accurate reflection of plasma heparin; however, calculated values rose to around 170% of anti-Xa values upon connection to bypass. By adjusting for this rise, for 95% of cases the calculated heparin concentration would vary between 0.60 and 1.65 times anti-Xa values. Without accounting for artificial prolongation or individual sensitivities, the ACT may give values between 0.8 and 3.0 times that indicated by the anti-Xa assay. When both individual heparin sensitivities and the effects of bypass are considered, the ACT may provide a more suitable indication of heparin levels; however, typical use may overestimate heparin up to threefold.

Postoperatively administered aprotinin or epsilon aminocaproic acid after cardiopulmonary bypass has limited benefit.

BACKGROUND: Intraoperative antifibrinolytic treatment with aprotinin and epsilon aminocaproic acid (EACA) has been shown to be effective prophylaxis in the reduction of excessive bleeding after cardiopulmonary bypass operations. This study investigated the effectiveness of both drugs when used as a postoperative treatment of patients showing early signs of increased bleeding. METHODS: In a double-blind, randomized study, 69 patients with chest drainage of 100 mL or more 1 hour after bypass were treated with aprotinin, EACA, or placebo. RESULTS: In the first 24 hours postoperatively, neither drug significantly reduced chest drainage or blood transfusion requirements compared with placebo. Median (interquartile) cumulative chest drainage volumes for the first 24 hours postoperatively for the aprotinin, EACA, and placebo groups were 525 (340, 750), 575 (450, 762), and 650 (550, 800) mL, respectively. Among the study patients, 4 undergoing valve operation and treated with aprotinin showed a trend toward less bleeding during the first 12 hours postoperatively compared with 5 valve operation patients who received placebo (p = 0.06). Among all patients, the treatment with aprotinin or EACA failed to reduce levels of D-dimer compared with placebo after treatment, indicating that fibrinolysis was not significantly inhibited. CONCLUSIONS: Aprotinin or EACA administered in the early postoperative period was ineffective in reducing postoperative bleeding with the exception of a small group of patients having valve operations in whom aprotinin treatment may have shown some benefit.

Epsilon-aminocaproic acid promotes the release of alpha2-antiplasmin during and after cardiopulmonary bypass.

This double-blind, randomized study compared the mechanisms by which low-dose aprotinin and epsilon-aminocaproic acid (EACA) inhibited fibrinolysis during cardiopulmonary bypass surgery. D-dimer levels during and after bypass were similar, indicating an equivalent inhibition of fibrinolysis. Effects on tissue plasminogen activator release were not associated with the inhibition of fibrinolysis by either drug. Treatment with EACA was associated with a substantial release of endogenous alpha2-antiplasmin, particularly 1 h after bypass. Compared with the aprotinin group, higher levels of the plasmin-alpha2-antiplasmin complex in the EACA group confirmed an increased inhibition of plasmin by alpha2-antiplasmin. In conclusion, it is hypothesized that EACA inhibited fibrinolysis by stimulating the release of the patients' own alpha2-antiplasmin.

Comparison of epsilon aminocaproic acid and low-dose aprotinin in cardiopulmonary bypass: efficiency, safety and cost.

BACKGROUND: In this study we compared the clinical efficiency, safety, and economic benefit of low-dose aprotinin with epsilon aminocaproic acid (EACA) in reducing bleeding after cardiopulmonary bypass operation. METHODS: In a double-blind, randomized study, 100 patients received low-dose aprotinin (2 x 10(6) kallikrein inhibitor units) or EACA (20 g). The surgical procedure was single- or double-valve replacement with or without coronary artery bypass grafts. RESULTS: Mediastinal chest drainage and transfusion requirements with both therapies were similar. There were no urgent reoperations to secure hemostasis in either group. Similar levels of D-dimer with both therapies indicate a similar inhibition of fibrinolysis. Release of troponin I was less in the low-dose aprotinin group 1 and 4 hours after bypass, although electrocardiographic measurements did not reflect this difference. Levels of S-100beta and neuron-specific enolase were similar with both therapies, confirming that there was no difference in the occurrence of any adverse neurologic events in either group. CONCLUSIONS: Low-dose aprotinin and EACA showed similar effects on the reduction of intraoperative and postoperative bleeding. The lower cost of EACA with no change in safety outcome suggests it is the preferred treatment.

Remifentanil and controlled hypotension; comparison with nitroprusside or esmolol during tympanoplasty.

PURPOSE: To determine whether remifentanil, combined with propofol, could induce controlled hypotension, reduce middle ear blood flow (MEBF) measured by laser-Doppler flowmetry, provide a "dry" operative field, and could be compared with nitroprusside or esmolol combined with alfentanil and propofol. METHODS: Thirty patients undergoing tympanoplasty and anesthetized with 2.5 mg x kg(-1) propofol iv followed by a constant infusion of 120 microg x kg(-1) x min(-1), were randomly assigned in three groups to receive either 1 microg x kg(-1) remifentanil iv followed by a continuous infusion of 0.25 to 0.50 microg x kg(-1) x min(-1), or nitroprusside iv, or esmolol iv combined for the latter two groups with alfentanil iv. RESULTS: Controlled hypotension was achieved at the target pressure of 80 mmHg within 107 +/- 16, 69 +/- 4.4, 53.3 +/- 4.4 sec for remifentanil, nitroprusside and esmolol respectively. MEBF decreased by 24 +/- 0.3, 22 +/- 3.3, 37 +/- 3% and preceded the decrease in SABP, within 30 +/- 6.1, 11.2 +/- 3.1, 15 +/- 2.8 sec for remifentanil, nitroprusside and esmolol respectively. Remifentanil, and nitroprusside decreased MEBF autoregulation less than esmolol (0.36 +/- 0.1, 0.19 +/- 0.2, -0.5 +/- 0.2). Controlled hypotension was sustained in all three groups throughout surgery, and the surgical field rating decreased in a range of 80% in all three groups. Nitroprusside decreased pH and increased PaCO2. There were no postoperative complications in any of the groups. CONCLUSIONS: Remifentanil combined with propofol enabled controlled hypotension, reduced middle ear blood flow and provided good surgical conditions for tympanoplasty with no need for additional use of a potent hypotensive agent.

Economic evaluation of high-dose and low-dose aprotinin therapy during cardiopulmonary bypass.

BACKGROUND: Aprotinin therapy is now widely used during cardiac surgery. This study examined the clinical and economic effectiveness of high-dose or low-dose aprotinin in comparison to placebo. METHODS: In a double blind, randomized study, three groups of 50 patients received high-dose aprotinin costing AUS$614 per patient (AUS$ = Australian dollars), low-dose aprotinin costing AUS$220 per patient or placebo. Resource use influenced by aprotinin therapy was measured. RESULTS: Both doses were effective in reducing chest drainage and postoperative transfusion requirements, high-dose being more effective than low-dose. Both doses reduced the rate of reoperations for hemostasis. A base case of statistically significant differences associated with the high-dose and low-dose aprotinin showed cost savings of AUS$77 and AUS$348 per patient, respectively. If the demonstrated less significant reductions in operating room and ward stay are included, these savings become AUS$463 and AUS$715, respectively. Alternately, if cross-matches are replaced by group-and-hold and cell savers are not used, the savings per patient would be AUS$196 and AUS$467, respectively. CONCLUSIONS: While high-dose aprotinin is clinically more effective than low-dose aprotinin, low-dose therapy demonstrates greater cost savings.

Aprotinin reduces blood loss after cardiopulmonary bypass by direct inhibition of plasmin.

The effectiveness and mechanism of aprotinin reduced bleeding after cardiopulmonary bypass surgery was studied in a double blind randomised study of 106 patients undergoing valve replacement surgery. Aprotinin therapy was associated with significant reduction in perioperative bleeding and postoperative blood transfusion requirements. Although initially tissue plasminogen activator (t-PA) activity was lower in the aprotinin than placebo group, as surgery proceeded this difference was reversed due to less plasminogen activator inhibitor-1 release in the aprotinin group. This indicates that aprotinin-mediated suppression of fibrinolysis as demonstrated by reduced D-dimer concentration was not related to t-PA. Furthermore, similar perioperative reduction of plasminogen levels in aprotinin and placebo groups indicated a similar degree of conversion of plasminogen to plasmin. However, less plasmin bound with alpha 2-antiplasmin in the plasma in the aprotinin group as it was already complexed with aprotinin where it remained protected from the natural inhibitor on the intact fibrin surface. The reduced fibrinolytic activity of the aprotinin group was thus brought about by the complexing of aprotinin with the plasmin which was bound to the fibrin surface.

Structural analysis of apolipoprotein A-I: limited proteolysis of methionine-reduced and -oxidized lipid-free and lipid-bound human apo A-I.

The domain structures of lipid-free and lipid-bound apolipoprotein A-I (apo A-I) containing reduced and oxidized methionines were analyzed by limited proteolysis. Lipid-free apo A-I is cleaved primarily in the extreme carboxy-terminus and, to a much lesser extent, in the central region of the protein between residues 115 and 136. Oxidation of methionines 112 and 148 to the corresponding sulfoxides in putative amphipathic helices 4 (P99-E120) and 6 (P143-A164), respectively, causes helices 1 (L44-G65), 2 (P66-S87), and 7 (P165-G186) to become susceptible to protease digestion. These results are consistent with a discrete, globular tertiary structure for the lipid-free protein minimally formed from amphipathic helices 1, 2, 4, 6, and 7. In distinct contrast to lipid-free apo A-I, lipid-bound apo A-I is most susceptible to cleavage in the extreme amino-terminus and, to a lesser extent, in both the central and carboxy-terminal regions. The observed cleavage pattern for the reduced lipid-bound protein supports the existence of many of the turns between helices predicted by sequence analysis of the lipid-bound protein. Methionine oxidation of lipid-bound protein results in a decreased protease susceptibility in the extreme amino-terminus and a concomitant increase in protease susceptibility in the central and carboxy-terminal regions. The results from methionine oxidation indicate the oxidation state of the protein is an important determinant in defining the conformation of both lipid-free and lipid-bound apo A-I.

Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation.

An amino-terminal deletion mutant (residues 1-43) of human apolipoprotein A-I (apo hA-I) has been produced from a bacterial expression system to explore the structural and functional role of these amino acids, encoded by exon 3, in apo hA-I. Lipid binding of apo delta (1-43)A-I and lipid binding of apo hA-I are very similar as assessed by surface activity, lipid association with palmitoyloleoylphosphatidylcholine (POPC) vesicles, and lipid association with plasma lipoproteins. Preliminary kinetic measurements appear to show that the reactivity of lecithin:cholesterol acyltransferase (LCAT) with the mutant is slightly decreased compared to wild-type apo hA-I. Collectively, these results indicate that the N-terminal region is not necessary for lipid binding or activation of LCAT. In contrast, there are significant structural differences between lipid-free apo delta (1-43)A-I and apo hA-I, as judged by denaturant-induced unfolding, binding of the fluorescent probe 1-anilinonaphthalene-8-sulfonate, surface balance measurements, and far- and near-ultraviolet circular dichroic spectroscopy. All spectral and physical measurements indicate apo delta (1-43)A-I has a folded, tertiary structure, although it is significantly less stable than that of apo hA-I. It is concluded that the N-terminal 43 residues are an important structural element of the lipid-free conformational state of apo hA-I, the absence of which induces a fundamentally different fold for the remaining carboxy-terminal residues, compared to those in native apo hA-I.

Preoperative platelet dysfunction increases the benefit of aprotinin in cardiopulmonary bypass.

BACKGROUND: This study was designed to determine the benefit of aprotinin therapy in reducing bleeding during and after cardiopulmonary bypass in patients with preoperative platelet dysfunction. Platelet function involvement in the mechanism by which aprotinin acts was also investigated. METHODS: In a double-blind, randomized study, patients received high-dose aprotinin (n = 54) or placebo (n = 52). Whole blood aggregation was measured preoperatively. Platelet function and activation in both groups were assessed intraoperatively and postoperatively at five times. RESULTS: Aprotinin significantly reduced perioperative bleeding and postoperative blood transfusion. Placebo-treated patients with reduced preoperative platelet aggregation bled more postoperatively, but aprotinin reduced the bleeding in patients with normal or reduced platelet function to similar levels. Any cardiopulmonary bypass-induced changes in platelet aggregation, platelet activation as measured by P-selectin expression, and von Willebrand factor antigen and function were similar in aprotinin-treated and placebo-treated groups. CONCLUSIONS: The mechanism by which aprotinin reduced bleeding was independent of any effect on platelet function. However, aprotinin produced a greater reduction in bleeding among patients whose condition was hemostatically compromised by preoperative platelet dysfunction.

Changes in plasminogen activator inhibitor-1 levels in non-small cell lung cancer.

Increased urokinase plasminogen activator (uPA) levels are increased in a number of malignancies and have been correlated with decreased disease-free interval and decreased overall survival. We have, therefore, examined components of this plasminogen activating system in patients with Non-Small Cell Lung Cancer (NSCLC). Levels of uPA, urokinase-plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) were measured semiquantitatively in paraffin sections of tumours from 147 patients with NSCLC. Immunohistochemically stained sections of tumour were allocated a score for stain intensity and results correlated to: survival; tumour stage(T); nodal stage(N); stage grouping (I to IIIb), survival status and sex. Increased levels of PAI-1 were associated with a decreased survival in squamous cell carcinoma (SCC) X2 = 5.72, p = 0.017 (n = 74). There was a significant positive relationship between PAI-1 levels and N-stage (p = < 0.05), presence of nodal metastases (p = < 0.05), stage grouping (p = < 0.01) and extent of disease (p = < 0.05) in the total group and the SCC subgroup, but not adenocarcinoma. There was a significant positive relationship between PAI-1 levels and T-stage (p = < 0.05) in the total group, and survival status (p = < 0.05) in the SCC subgroup alone. uPA and uPAR levels were not significantly associated with tumour staging or survival. We conclude that increased PAI-1 antigen levels may be associated with a decreased survival in patients with SCC.

A proposed model to monitor heparin therapy using the concentrated thrombin time which allows standardisation of reagents and improved estimation of heparin concentrations.

The concentrated thrombin time (CTT), a thrombin time performed with a high concentration of thrombin, was evaluated as an alternative to the activated partial thromboplastin time (APTT) for monitoring of heparin therapy. Forty-nine plasmas from patients receiving unfractionated heparin therapy were tested. It was first demonstrated that CTTs using three commercial reagents could be standardised against CTTs performed with a reference reagent, MRC reagent 66/305. For comparison, APTTs were performed on the plasmas. As a benchmark of the degree of heparinisation, the heparin concentration of the plasmas was determined by chromogenic anti-IIa heparin assays, therapeutic range being 0.2-0.4 units/ml. The optimal relationships of the CTTs and APTT with the heparin concentration were established. These were used to predict the heparin concentrations of the plasmas from the results of the APTT, CTT performed with the reference reagent, and transformed CTT performed with each of the three commercial reagents. In predicting the assayed plasma heparin concentrations, the accuracy of the APTT was only 53%, while the CTT was from 78 to 82%. The CTT can be standardised and, subject to results of clinical trials, could provide an improved method of monitoring heparin therapy.

The role of von Willebrand factor in haemostasis and blood loss during and after cardiopulmonary bypass surgery.

Excessive perioperative and postoperative bleeding continue to complicate cardiopulmonary bypass surgery (CPB). In this study we measured the von Willebrand factor antigen (vWF:Ag), collagen binding assay (CBA) and ristocetin cofactor assay (RiCo) in 52 patients undergoing CPB. The collagen binding assay employs the affinity of the high-molecular-weight multimers of vWF to collagen and was used in this study to demonstrate differences in the multimeric composition of vWF during and after CPB. The observed values for the vWF:Ag, CBA and RiCo were correlated to the amount of postoperative bleeding. Both the preoperative vWF:Ag and the CBA showed significant negative correlation with postoperative blood loss (r = -0.3046, P < 0.05 and r = -0.3228, P < 0.05 respectively). Higher blood loss figures correlated with lower vWF:Ag, CBA and RiCo during and after surgery with the strongest correlation 1 h post-op between both vWF:Ag and CBA and actual blood loss (r = -0.5061, P < 0.001 and r = -0.4942, P < 0.001 respectively). The strong negative correlations between vWF:Ag, CBA and RiCo before, during and after CPB and blood loss data verify the important role of vWF in primary haemostasis. Of particular note, the negative correlations between preoperative levels of vWF:Ag, CBA and postoperative blood loss provide valuable insight into another possible mechanism of excessive bleeding post CPB.