A 3-O-methylated mannogalactan from Pleurotus pulmonarius: structure and antinociceptive effect.

A polysaccharide (Mw 2.39x10(4)g/mol) was extracted with cold water from the basidiomycete Pleurotus pulmonarius, and its antinociceptive and anti-inflammatory properties were evaluated. It was a mannogalactan (MG), whose structure was characterized using mono- and two-dimensional NMR spectroscopy, methylation analysis, and a controlled Smith degradation. It had a main chain of (1-->6)-linked alpha-D-galactopyranosyl and 3-O-methyl-alpha-D-galactopyranosyl units, both of which are partially substituted at O-2 by beta-D-mannopyranosyl non-reducing ends. The MG was tested for its effects on the acetic acid-induced writhing reaction in mice, a typical model for inflammatory pain, causing a marked and dose-dependent inhibition of the nociceptive response, with ID50 of 16.2 (14.7-17.7)mg/kg and inhibition of 93+/-3% at a dose of 30mg/kg. An inflammatory response was not inhibited.

Site-specific interactions of copper(II) ions with heparin revealed with complementary (SRCD, NMR, FTIR and EPR) spectroscopic techniques.

The interactions between Cu(II) ions and heparin were investigated using several complementary spectroscopic techniques. NMR indicated an initial binding phase involving specific coordination to four points in the structure that recur in slightly different environments throughout the heparin chain; the carboxylic acid group and the ring oxygen of iduronate-2-O-sulfate, the glycosidic oxygen between this residue and the adjacent (towards the reducing end) glucosamine and the 6-O-sulfate group. In contrast, the later binding phase showed little structural specificity. One- and two-dimensional correlated FTIR revealed that complex out of phase (asynchronous) conformational changes also occurred during the titration of Cu(II) ions into heparin, involving the CO and N-H stretches. EPR demonstrated that the environments of the Cu(II) ions in the initial binding phase were tetragonal (with slightly varied geometry), while the later non-specific phases exhibited conventional coordination. Visible spectroscopy confirmed a shift of the absorbance maximum. Titration of Cu(II) ions into a solution of heparin indicated (both by analysis of FTIR and EPR spectra) that the initial binding phase was complete by 15-20 Cu(II) ions per chain; thereafter the ions bound in the non-specific mode. Hetero-correlation spectroscopy (FTIR-CD) improved resolution and assisted assignment of the broad CD features from the FTIR spectra and indicated both in-phase and more complex out of phase (synchronous and asynchronous, respectively) changes in interactions within the heparin molecule during the titration of Cu(II) ions.

Determination of thorium isotopes in mineral and environmental water and soil samples by alpha-spectrometry and the fate of thorium in water.

A method has been developed for determination of thorium isotopes in water and soil samples by alpha-spectrometry. After fusion with Na(2)CO(3) and Na(2)O(2) at 600 degrees C, soil samples were leached with HNO(3) and HCl. Thorium in water sample or in soil leaching solution was coprecipitated together with iron (III) as hydroxides and/or carbonates at pH 9 with ammonia solution, separated from uranium and other alpha-emitters by a Microthene-TOPO (tri-octyl-phosphine oxide) chromatographic column, electrodeposited on a stainless steel disk, and measured by alpha-spectrometry. The method was checked with two certified reference materials supplied by the IAEA, and reliable results were obtained. The detection limits of the method for water (soil) samples are 0.44 microBq l(-1) (0.070 Bq kg(-1)) for (232)Th, 0.80 microBq l(-1) (0.13 Bq kg(-1)) for (230)Th and 1.0 microBq l(-1) (0.16 Bq kg(-1)) for (228)Th, respectively, if 100 l of water (0.50 g) for each sample are analysed. A variety of water or soil samples were analysed using this procedure and giving average thorium yields of 75.5+/-14.2% for water and 93.4+/-4.5% for soil. The obtained concentrations of thorium isotopes in water samples are in the range of 0.0007-0.0326 mBq l(-1) for (232)Th,

The effect of increasing the sulfation level of chondroitin sulfate on anticoagulant specific activity and activation of the kinin system.

Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.

Midazolam and pethidine versus propofol and fentanyl patient controlled sedation/analgesia for upper gastrointestinal tract ultrasound endoscopy: a prospective randomized controlled trial.

BACKGROUND AND OBJECTIVES: The aim of this prospective, randomized study was to compare the standard regimen of midazolam and pethidine administered by the gastroenterologist versus patient controlled sedation with a propofol-fentanyl mixture during upper gastrointestinal tract endoscopic ultrasonography. Our primary end-points were patient satisfaction and patient cooperation assessed by endoscopist. METHODS: Fifty-four consecutive patients, undergoing endoscopic ultrasonography, received sedation with midazolam and pethidine (Group M: n=27) or propofol and fentanyl (Group P: n=27). Group M: pethidine 0.7mg/kg midazolam 0.04mg/kg before examination; boluses of same drugs if the sedation was insufficient plus a sham patient controlled sedation analgesia; Group P: propofol 17mg plus fentanyl 15microg before examination and a patient controlled sedation analgesia pump containing 170mg propofol plus 150microg fentanyl injecting 0.5ml every time the patient pressed the button (no "lock out"). Boluses of 1ml of the same mixture if the sedation was insufficient. RESULTS: Group M: mean dosage of pethidine and midazolam 88.6 and 5mg, respectively. Group P: mean dosage of propofol and fentanyl 119.7mg and 106microg, respectively. Both groups were similar for duration and difficulty of the procedure, the grade of sedation (Observer's Assessment of Alertness/Sedation Score) and judgement by endoscopist and patient about cooperation and satisfaction. The only difference between groups was about the extra boluses administered during the procedure. CONCLUSION: This study demonstrated that a patient controlled sedation analgesia with propofol and fentanyl is an effective and safe technique for upper gastrointestinal tract endoscopic ultrasonography procedures and results in a high level of satisfaction both for patients and operator.

Target-controlled infusion during monitored anesthesia care in patients undergoing EUS: propofol alone versus midazolam plus propofol. A prospective double-blind randomised controlled trial.

BACKGROUND: It has been speculated that midazolam may be effective in reducing the required dose of propofol during sedation. AIM: To evaluate the sparing effect of midazolam during target-controlled propofol infusion. METHODS: Two hundred-seventy patients undergoing upper endoscopic ultrasound were randomised to receive sedation with propofol plus placebo (group A) or plus midazolam (group B). Outcome parameters were the procedure duration, the discharge time and the satisfaction of patients, operator and nurse about the quality of sedation. RESULTS: The mean propofol dose administered was 364+/-207 mg in group A and 394+/-204 mg in group B. Mean procedure duration (group A: 32+/-17 min, group B: 35+/-22 min) and discharge time (group A: 39+/-30 min, group B: 38+/-24 min) were similar in both groups. No severe complications were observed. The quality of sedation was judged satisfactory for all patients by both the endoscopist and the nurse assistant without any difference between the two groups. No patient remembered the procedure or reported it as unpleasant. CONCLUSIONS: Target-controlled propofol infusion provides safe and effective sedation; premedication with low dose of midazolam does not reduce the total amount of propofol administered. Further studies are needed to compare propofol alone with propofol co-administered with opioid.

A comparative study of low-density lipoprotein interaction with glycosaminoglycans.

The association between low-density lipoprotein (LDL) and a series of well characterized dermatan and chondroitin sulfates has been investigated by means of the fluorescence anisotropy technique with competition experiments using a fluorescein-labeled high LDL-affinity heparin fraction as a reference. Preparations of glycosaminoglycan (GAG) with sulfation degrees varying over a wide range, as obtained by fractionation or by chemical modification, were chosen for this study. The influence of chain length, which had been found sizeable in a former study of heparin affinity for LDL, was taken into account with an empirical correction of dissociation constants. After this correction, a linear relationship was found between the logarithm of dissociation constants and the number of sulfate groups per disaccharide unit, ns, both for dermatan and chondroitin sulfates, and for heparins. At comparable ns values, however, dermatan sulfates and heparins, which contain L-iduronic acid in their backbone, show higher LDL-affinity than chondroitin sulfates, which contain only D-glucuronic acid. Though confirming a non-specific, predominantly electrostatic interaction between GAGs and LDL, these results indicate modulation of LDL affinity by the polysaccharide backbone.

Specific antibodies to diphtheria toxin and type 6A pneumococcal capsular polysaccharide induced by a model of semi-synthetic glycoconjugate antigen.

A molecular model of a carbohydrate-protein conjugate is described, involving the non-toxic mutant protein CRM197, serologically related to the diphtheria toxin, covalently bound to a characterized oligosaccharide derived from the molecular structure of type 6A pneumococcal capsular polysaccharide. Physicochemical and immunochemical characteristics of this oligosaccharide-protein conjugate were consistent with a molecule showing a molar carbohydrate/protein ratio of 8, an av. mol. wt of 75,000, and retention of complete immunochemical identity when tested towards the homologous antisera. The immunological characteristics obtained after immunization of 2 animal models showed a high immunogenicity of the glycoconjugate specifically directed towards diphtheria toxin and the type 6A pneumococcal capsular polysaccharide.

High resolution NMR spectroscopy of physiological fluids: from metabolism to physiology.

High resolution NMR spectroscopy of physiological fluids provides quantitative, qualitative and dynamic information on the metabolic status of the interstitial and plasma compartments under a variety of pathophysiological conditions. The simultaneous detection and quantitation by NMR spectroscopy of numerous compounds of the intermediary metabolism offers a new insight in the understanding of the milieu intérieur. NMR spectroscopy of physiological fluids offers a unique way to define and monitor the global metabolic homeostasis in humans. The development of this analytical approach is still limited by the scarcity of pluridisciplinary teams able to fully exploit the wealth of information present on the NMR spectrum of a fluid. While application in pharmacology and toxicology is already established, the main areas of current development are cancer, hereditary metabolic disorders, organ transplantation and neurological diseases.

Antithrombotic and bleeding effects of a low molecular weight heparin fraction.

Low molecular weight (LMW) heparin prevents venous thrombosis by potentiating the inhibition of coagulation factor Xa. Heparin, however, has other biological properties whose role in the prevention of thrombosis is still unknown. The aim of our study was to compare the antithrombotic activity of a LMW heparin and its parent molecule in an attempt to understand better the mechanism and structural requirements for heparin's antithrombotic effect. We studied a preparation of an unfractionated pig mucosal heparin pure by any accepted criteria (electrophoresis in various systems, conductimetric titration and NMR spectra) and a LMW heparin fraction obtained from the former by fractional precipitation with ethanol. Both heparins completely prevented thrombus formation in an experimental model of stasis-induced venous thrombosis in rats. When administered intravenously to rats, the unfractionated heparin had an ex vivo anti-Xa/APTT ratio of 1.67, versus 6.60 of the LMW heparin fraction. Unexpectedly, both heparins induced a significant prolongation of tail bleeding time, performed by two different techniques, the "transection" (mostly exploring blood clotting) and the "template" (exploring the platelet/vessel wall interactions). This study suggests that, beside anticoagulation, other effects may play a role in both the antithrombotic and haemorrhagic effects of some heparins and LMW heparin fractions.

"Supersulfated" heparin fragments, a new type of low-molecular weight heparin. Physico-chemical and pharmacological properties.

A new type of low-molecular-weight heparin (ss-LMW-H) was prepared (by controlled depolymerization and concurrent sulfation of heparin with a mixture of sulfuric and chlorosulfonic acid), to test the influence of extra-sulfate groups on biological properties of heparin fragments. The fragments had an average molecular weight ranging from 5000 to 10,000, a sulfate-to-carboxyl molar ratio of 2.8-3.1, and electrophoretic mobilities and NMR spectra distinctly different from those of the parent heparins. Depolymerization with oversulfation reduced the anticoagulant activity of heparin (ex vivo, in rats) much more than depolymerization alone, to about 10% of the original APTT and 25-30% of the original a.Xa units. By contrast, the antithrombotic activity (venous stasis model, in rats) was still comparable to that of heparin, and bleeding times were not significantly increased. The lipasemic (lipoprotein-lipase-releasing) activity of ss-LMW-H fragments was more than twice that of heparin. Results are discussed in terms of contribution of charge-density effects to different activities and to different mechanisms for the same activity of heparin.

Effect of nasal pressure support ventilation and external PEEP on diaphragmatic activity in patients with severe stable COPD.

Nasal pressure support ventilation (NPSV) has been shown to be useful in the treatment of acute and chronic pulmonary failure. However, little is known about respiratory muscle activity during NPSV in stable patients with COPD. The aim of this study was to test the effect of two levels (10 and 20 cm H2O) of NPSV on diaphragmatic activity, in a group of seven stable, severe COPD patients (FEV1 20 percent +/- 7 of pred, FEV1/FVC 35 percent) with hypercapnic respiratory insufficiency. Since these patients had an intrinsic PEEP (PEEPi) of 2.6 +/- 1.3 cm H2O, we also investigated the effect of adding 5 cm H2O external PEEP (PEEPe) during NPSV. Blood gases, ventilatory pattern by inductive plethysmography, integrated electromyogram of the diaphragm (Edi), transdiaphragmatic pressure (Pdi), and the diaphragmatic pressure time product (PTPdi) were recorded during randomized 15-min runs of both levels of NPSV with and without the addition of PEEPe. Minute ventilation did not change with the application of NPSV, but a significant decrease in breathing frequency with a parallel increase in tidal volume was observed, so that blood gas determinations improved at the higher levels of support. A marked statistically significant reduction in diaphragmatic activity, as assessed by a decrease in Pdi swings, PTPdi, and Edi, was detected at the levels of 10 and 20 cm H2O; a further significant decrease in these values was observed when PEEPe was added. PEEPi decreased significantly only with the application of PEEPe, resulting in a small increase in end-expiratory lung volume. We conclude that NPSV improves diaphragmatic function in patients with severe stable COPD; this effect may be enhanced by the applications of external PEEP.

Tranexamic acid compared with high-dose aprotinin in primary elective heart operations: effects on perioperative bleeding and allogeneic transfusions.

OBJECTIVE: Since excessive fibrinolysis during cardiac surgery is frequently associated with abnormal perioperative bleeding, many authors have advocated prophylactic use of antifibrinolytic drugs to prevent hemorrhagic disorders. We compared the effects of tranexamic acid (a synthetic antifibrinolytic drug) with aprotinin (a natural derivative product with antifibrinolytic properties) on perioperative bleeding and the need for allogeneic transfusions. METHODS: In a single-center prospective randomized unblinded trial, 1040 consecutive patients undergoing primary, elective cardiac operations with cardiopulmonary bypass received either high-dose aprotinin or tranexamic acid. The aprotinin group (518 patients) received 280 mg in 20 minutes before the skin incision, 280 mg in the priming solution of the extracorporeal circuit, and a continuous infusion of 70 mg/h throughout the operation. The tranexamic acid group (522 patients) received 1 g in 20 minutes before the skin incision, 500 mg in the priming solution of the extracorporeal circuit, and a continuous infusion of 400 mg/h during the operation. Postoperative bleeding, perioperative transfusions, and hematologic variables were evaluated at fixed times. Postoperative thrombotic complications, intubation time, intensive care unit stay, and hospital stay were recorded. RESULTS: Postoperative bleeding was similar in the 2 groups: aprotinin 250 mL (150-400 mL) versus tranexamic acid 300 mL (200-450 mL) (median and 25th-75th quartiles), median difference of 50 mL (95% confidence intervals, 0-50 mL). The number of transfusions and the outcome did not differ. CONCLUSIONS: Tranexamic acid and aprotinin show similar clinical effects on bleeding and allogeneic transfusion in patients undergoing primary elective heart operations. Since tranexamic acid is about 100 times cheaper than aprotinin, its use is preferable in this type of patient.

Emergency endovascular treatment of a life-threatening hemorrhage from traumatic rupture of the left extracranial vertebral artery.

Hemorrhagic complications from transection of cervical arteries in blunt traumas are rare. We report a case of potentially fatal hemorrhage from rupture of the left vertebral artery in a closed trauma, successfully treated by endovascular injection of glue. Endovascular embolization may be considered as an alternative to surgical exploration in the treatment of traumatic lesions of vertebral arteries.

Respiratory mechanics in anesthetized paralyzed humans: effects of flow, volume, and time.

The effects of inspiratory flow rate and inflation volume on the resistive properties of the total respiratory system were investigated in 16 anesthetized paralyzed humans by using the technique of rapid airway occlusion during constant flow inflation. This allowed measurement of the intrinsic resistance (Rmin,rs) and of the effective additional resistance (delta Rrs) as the result of viscoelastic pressure dissipations within the pulmonary and chest wall tissues. We observed that 1) at fixed inflation volume, Rmin,rs increased linearly with increasing flow although delta Rrs decreased according to an exponential function; 2) at fixed inflation flow, Rmin,rs decreased with increasing inflation volume although there was a concomitant increase in delta Rrs. This behavior could be explained in terms of a spring-and-dashpot model incorporating 1) the standard resistance and elastance and 2) a spring-and-dashpot in parallel with standard elastance, reflecting the stress adaptation units within the thoracic tissues.

Pulmonary and chest wall mechanics in anesthetized paralyzed humans.

Pulmonary and chest wall mechanics were studied in 18 anesthetized paralyzed supine humans by use of the technique of rapid airway occlusion during constant-flow inflation. Analysis of the changes in transpulmonary pressure after flow interruption allowed partitioning of the overall resistance of the lung (RL) into two compartments, one (Rint,L) reflecting airway resistance and the other (delta RL) representing the viscoelastic properties of the pulmonary tissues. Similar analysis of the changes in esophageal pressure indicates that chest wall resistance (RW) was due entirely to the viscoelastic properties of the chest wall tissues (delta RW = RW). In line with previous measurements of airway resistance, Rint,L increased with increasing flow and decreased with increasing volume. The opposite was true for both delta RL and delta RW. This behavior was interpreted in terms of a viscoelastic model that allowed computation of the viscoelastic constants of the lung and chest wall. This model also accounts for frequency, volume, and flow dependence of elastance of the lung and chest wall. Static and dynamic elastances, as well as delta R, were higher for the lung than for the chest wall.

Tranexamic acid in off-pump coronary surgery: a preliminary, randomized, double-blind, placebo-controlled study.

BACKGROUND: We evaluated the hemostatic effects of tranexamic acid, a synthetic antifibrinolytic drug, in patients undergoing beating-heart coronary surgery. METHODS: Forty consecutive patients were in a double-blind manner, prospectively randomized into two groups: 20 patients received tranexamic acid (bolus of 1 g before skin incision, followed by continuous infusion of 400 mg/hr during surgery), and 20 patients received saline. As primary outcomes, bleeding and allogeneic transfusions were considered. D-dimer and fibrinogen plasma levels were also evaluated to monitor the activation of fibrinolysis. Major postoperative thrombotic events, as a potential consequence of antifibrinolytic treatment, were recorded. RESULTS: The treatment group had significantly lower postoperative bleeding (median [25th to 75th percentiles]: 400 mL [337 to 490 mL] vs 650 ml [550 to 862 mL], p < 0.0001), lower need for allogeneic blood products (1,200 vs 5,300 mL, p < 0.001), and lower postoperative D-dimer plasma levels. No postoperative thrombotic complications were observed in either group. CONCLUSIONS: In this initial series of patients undergoing off-pump coronary surgery, tranexamic acid appears to be effective in reducing postoperative bleeding and the need for allogeneic blood products.

Hemostatic effects of aprotinin, tranexamic acid and epsilon-aminocaproic acid in primary cardiac surgery.

BACKGROUND: The effects of epsilon-aminocaproic acid (EACA) and tranexamic acid (TA) on bleeding and allogeneic transfusions, and the cost of pharmacological and transfusional treatment were compared to aprotinin (AP). METHODS: We randomized 210 patients subjected to elective cardiac surgery. Of these, 68 patients received EACA (a bolus of 5 g, an infusion of 2 g/h, and 2.5 g in the priming), 72 patients received TA (a bolus of 1 g, an infusion of 400 mg/h, and 500 mg in the priming), and 70 patients received AP (a bolus of 280 mg, an infusion of 70 mg/h, and 280 mg in the priming). Postoperative blood loss and homologous transfusions were collected and the cost of pharmacological treatment and homologous transfusions were calculated. RESULTS: Bleeding but not allogeneic transfusions was significantly higher in the EACA group (467+/-234 versus TA, 311+/-231 versus AP, 283+/-233; p < 0.001). Costs of pharmacological and transfusional treatment were significantly lower in the TA group ($58.10+/-$105.10) versus the EACA group ($100.70+/-$158.60) versus the AP group ($432.60+/-$118.70) (p < 0.0001). CONCLUSIONS: Compared to AP, TA has the same effects on bleeding and transfusions, but with a significant reduction of costs. Patients treated with EACA showed a significantly higher postoperative bleeding with an increased trend of transfusion requirement.

Magnetic bead protamine-linked microtiter assay for detection of heparin using iodinated low-molecular-mass heparin-tyramine.

We have developed a competitive heparin binding assay employing protamine-coated magnetic beads for detection and measurement of heparin. The assay utilizes 125-iodine specifically bound to newly synthesized low-molecular-mass (LMM) heparin-tyramine. The tracer was stable over a period of 3 weeks, as demonstrated by gel filtration chromatography. The protamine-coated beads were found to be stable over at least two months. The heparin-tyramine bead assay had in buffer a lower detection limit of 0.04 microgram/ml and in plasma of 0.23 microgram heparin/ml. 50% binding was obtained at 0.7 microgram/ml and 20% binding at 4 micrograms/ml in plasma. The within assay coefficient of variation ranged from 9 to 28% for unfractionated, high molecular mass (HMM) heparin and from 12 to 15% for LMM-heparins in buffer system and in plasma. Various heparin fractions displaced the tracer from the protamine-coated magnetic beads to different extents. The validity of the assay was proven after intravenous administration of unfractionated and LMM-heparin in man. The elimination rate was similar using the heparin-tyramine bead assay compared with the anti-factor Xa coagulation assay. After intravenous dosing of LMM-heparin the maximal concentration was lower using the heparin-tyramine bead assay compared with the anti-factor Xa coagulation assay. The bead assay was found to be reproducible, valid, and rapid for measurement of the concentration of heparin preparations in purified systems and for HMM-heparin in plasma. Measurement of the concentration of LMM-heparin in plasma has a high coefficient of variation using the binding assay.