Treatment of systemic inflammatory response syndrome by push-pull powdered sorbent pheresis: a Phase 1 clinical trial.

An FDA-approved Phase 1 feasibility study was performed in two centers to determine the safety of the BioLogic-DTPF (detoxifier/plasma filter) system for the treatment of patients with systemic inflammatory response syndrome (SIRS). This device combines hemodiabsorption (dialysis of blood against powdered sorbents with the BioLogic-DT system) with push-pull sorbent-based pheresis (the PF add-on module). Eight adult ICU patients with both SIRS and multiple organ failure participated in the study. One 6 h treatment was planned for each patient with powdered charcoal as sorbent for 4 patients and a combination of charcoal/silica in the PF sorbent bag for 4 patients. The treatments appeared to have no negative effects in 7 patients, but 1 patient died during treatment due to progressive cardiac failure. Sepsis was resolved in 5 of the 8 patients. However, there were only 2 long-term survivors of the group. The addition of the PF module should improve the chemical function of the BioLogic-DT by allowing removal of protein-bound toxins such as cytokines. The selected patients tolerated treatment by the DTPF system well, but proof of benefit of the device remains to be proven in a Phase 2 clinical trial with randomized controls.

Effect of hemodiabsorption and sorbent-based pheresis on amino acid levels in hepatic failure.

Changes in plasma amino acid concentrations were measured in patients with hepatic failure during extracorporeal hemodiabsorption (using the Liver Dialysis Unit, "the Unit") or hemodiabsorption plus sorbent-based pheresis treatment (using the Liver Dialysis Plasmafilter Unit, "the PF-Unit") Systems. Eight patients with hepatic failure, grade 3 or 4 encephalopathy, elevated bilirubin and/or creatinine levels and respiratory or renal failure were treated for 1-3 days with the Unit alone. Three of these were also treated with the Unit containing 10 g of BCAA in the sorbent suspension. Four patients with hepatic failure treated with the PF Unit also had 10 g of branched chain amino acid (BCAA) added to the sorbents of the Unit portion of this device. Pre- and post-plasma samples were drawn and high performance liquid chromatography (HPLC) was used to separate and detect amino acids in the plasma. Both the Unit and the PF-Unit have the capability to selectively remove various amino acids, especially aromatic amino acids (AAA). The pre-treatment amino acid profiles of plasma were typical for hepatic failure, with abnormally high levels of phenylalanine, tyrosine, tryptophan, and methionine and decreased levels of valine, leucine and isbolucine. The average pre-treatment Fischer ratio (BCAA/AAA) for both Unit and PF-Unit patients was 1.43 (+/- 0.58). Treatments by both systems resulted in an increase of BCAA levels in blood and concomitant decrease of AAA levels, with an average Fischer ratio improvement of 30-38% for the Unit and PF-Unit without BCAA. The Fischer ratio improved by 90% (average) for the Unit with BCAA. Levels of many other amino acids (such as alanine, glycine, proline or lysine) increased during both Unit and PF-Unit treatments. The removal of strongly protein-bound toxin and amino acids such as tryptophan and sulphydryl amino acids was more effective by the PF-Unit. Both the Unit and the PF-Unit have the unique capability to remove toxic aromatic amino acids while increasing BCAA levels in patient. The increase in many amino acid levels may be related to the removal of toxins that interfere with normal amino acid metabolism. The addition of the PF module improves the removal of bilirubin and similarly protein-bound chemicals. Changes in amino acid profiles by the Unit and the PF-Unit contrast markedly with other extracorporeal devices.

Push-pull sorbent-based pheresis and hemodiabsorption in the treatment of hepatic failure: preliminary results of a clinical trial with the BioLogic-DTPF System.

The BioLogic-DTPF System combines hemodiabsorption (the BioLogic-DT System with dialysis against powdered sorbent) with push-pull sorbent-based pheresis (the BioLogic-PF System with powdered sorbent surrounding plasma filters). At blood flow rates of 200 ml/min, the system clears creatinine and aromatic amino acids at 120-160 ml/min, unconjugated bilirubin at 20-40 ml/min, and cytokines at 15-25 ml/min. This article outlines a study of the DTPF System in treatment of patients with hepatic failure with Grade 3 or 4 encephalopathy and respiratory and kidney insufficiency. Treatment appeared to be safe, and there are no significant hematologic changes. Physiologic changes include improved blood pressure and encephalopathy and stable urine output. Chemical changes include decrease in plasma levels of bilirubin, aromatic amino acids, ammonium, creatinine, and interleukin-3 (IL-1beta). The BioLogic-DT System is now marketed for treatment of acute hepatic failure with encephalopathy. The BioLogic-DTPF System adds the capability of removing bilirubin and other strongly protein-bound toxins from treated patients and may be of clinical benefit in management of patients with the most severe hepatic failure and encephalopathy.

Push-pull sorbent-based pheresis treatment in an experimental canine endotoxemia model: preliminary report.

The Biologic-DTPF System (DTPF), an extracorporeal blood treatment device with potential to treat sepsis, was tested in a preliminary study using a canine endotoxemia model. Six dogs were used and they formed four treatment groups, as control group (n=1) and three groups based on the type of sorbent present in the plasma filter (PF) system: sham treatment with no sorbent (n=1), charcoal as sorbent (n=2), and charcoal/silica as sorbent ("silica" group, n=2). Cardiodynamic data were recorded before treatment and every 30 minutes, and blood samples were collected to determine blood chemistry and to detect the levels of endotoxin and selected plasma cytokines: interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF). The dogs were given Escherichia coli endotoxin (2 mg/kg) as an intravenous drip (extended over a period of 30 minutes). Thirty minutes after the end of infusion all animals except the control were treated with the DTPF system for four hours. To determine the effect of treatment, data collected at one hour from the initiation of treatment until the end of treatment were compared between control and treated dogs. The endotoxin levels in the control dog were higher (P < 0.05) than other groups. The control dog had lower levels of TNF than other groups. The control dog had similar levels of IL-1 (P > 0.05) and higher levels (P < 0.05) at 4 hours into treatment compared to other groups. The control dog had similar levels of IL-6 as other groups (P > 0.05). In the control dog, the mean arterial pressure (MAP) fell and then remained low but stable at 1-4 hours. The charcoal group had lower MAP than the control dog at 1-4 hours (P < 0.05). The silica group had higher MAP levels similar to the control dog. After treatment, the control dog had higher (P < 0.05) values of hematocrit, hemoglobin, calcium, potassium, and albumin compared to the treated groups. As expected for a system removing plasma during sepsis, the DTPF System had some adverse effects on the physiologic status of the dogs, especially when loaded with charcoal sorbent only. The findings of the present study suggest that the filters are capable of eliminating endotoxin and there is some evidence of cytokine removal. Although the charcoal dogs did poorly, addition of silica to the sorbent offset any negative effects. Further work is underway to improve the efficiency of the system, primarily to enhance the capacity of the sorbents for cytokines. A more realistic canine sepsis model with mortality after several days (the Escherichia coli- infected intraperitoneal clot) will also be considered in future studies.

Cytokines and endotoxin removal by sorbents and its application in push-pull sorbent-based pheresis: the BioLogic-DTPF System.

The BioLogic-DTPF System (DTPF) combines the Biologic-DT hemodiabsorption system (DT) in series with the Biologic PF push-pull pheresis system (PF) in which PF membranes separate plasma for direct contact between plasma proteins and the sorbents. Preliminary studies conducted in bovine serum albumin (BSA) solution and in bovine plasma allowed charcoal and silica to be evaluated as adsorbents for the PF module. Equilibrium binding experiments in BSA showed a high capacity of cytokine (IL-1 beta, TNF alpha) binding by powdered charcoal, 70-90 ng/g. Kinetic binding studies in bovine plasma revealed relatively quick adsorption of IL-1 beta and IL-6 by charcoal with the capacity range of 1.2-2.0 ng/g for tested cytokines (IL-1 beta and TNF alpha). Further laboratory studies with plasma have shown that powdered silica has an even greater binding capacity, up to 13 ng/g for TNF alpha depending upon particle size, and more rapid binding for all tested cytokines than powdered charcoal. Cholestyramine is a more efficient sorbent for removal of endotoxin than either charcoal or silica. In vitro tests using whole blood have demonstrated that the DTPF, with powdered charcoal as the sorbent, clears cytokines (TNF alpha, IL-1 beta, and IL-6) at 12.6-23.4 ml/min, bilirubin at 17.8-34.7 ml/min, and creatinine at 53.6-82.6 ml/min. The removal of some cytokines during the first clinical trial is also discussed.

Systemic inflammatory response syndrome treatment by powdered sorbent pheresis: the BioLogic-Detoxification Plasma Filtration System.

Systemic inflammatory response syndrome (SIRS) is one of the most common causes of death in intensive care unit patients. The detoxification plasma filtration (DTPF) system (HemoCleanse, Inc., West Lafayette, IN) combines the DT hemodiabsorption system in series with a push-pull pheresis PF system (a suspension of powdered sorbents surrounding 0.5 microm plasma filter membranes). Bidirectional plasma flow (at 80-100 ml/min) across the PF membranes provides direct contact between plasma proteins and powdered sorbents, as well as clearance of cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) at a rate of 15-25 ml/min, without evidence of saturation for 90 minutes. In a U.S. Food and Drug Administration approved study we treated eight patients with SIRS and organ failure with a single DTPF treatment, using powdered charcoal as sorbent in four patients and powdered charcoal and silica in four patients. Treatments proceeded for 6 hours with proper heparin anticoagulation (activated clotting time 250-300 sec) and appeared safe. All patients improved during the treatments and each had increased blood pressure and decreased need for pressor agents. Plasma cytokine levels stabilized or decreased during treatment and were significantly lower the morning after treatment. Multiple organ dysfunction (MOD) and Acute Physiology Chronic Health Evaluation II scores and organ function gradually improved in most patients, and two patients survived for more than 28 days and two for more than 14 days. The DTPF System may prove beneficial in treatment of patients with sepsis.

Uncovering a calcium-regulated membrane-binding mechanism for soybean lipoxygenase-1.

Lipoxygenases catalyze the biosynthesis of leukotrienes, lipoxins, and other lipid-derived mediators that are involved in a wide variety of pathophysiological processes, including inflammation, allergy, and tumorigenesis. Mammalian lipoxygenases are activated by a calcium-mediated translocation to intracellular membranes upon cell stimulation, and cooperate with cytosolic phospholipase A2 at the membrane surface to generate eicosanoids. Although it has been documented that plant cell stimulation increases intracellular Ca2+ concentration and activates cytosolic phospholipase A2, followed by lipoxygenase-catalyzed conversion of the liberated linolenic acid to jasmonic acid, no evidence is available for Ca2+-regulated membrane binding and activity of plant lipoxygenases. Plant lipoxygenases, unlike their mammalian counterparts, are believed to function independently of calcium or membranes. Here we present spectroscopic evidence for a calcium-regulated membrane-binding mechanism of soybean lipoxygenase-1 (L-1). Both calcium and membrane binding affect the structure and the mode of action of L-1. Free L-1 in solution is less accessible to the polar solvent and converts linoleic acid to conjugated dienes, whereas surface binding increases solvent accessibility and stimulates conjugated ketodiene production. Calcium exerts a biphasic effect on the structure and activity of L-1. Our results uncover a new regulatory mechanism for plant lipoxygenases and delineate common features in animal and plant cell signaling pathways.

Amino-acid composition of pyruvate kinase M2 isoenzyme variants from rat liver and Morris hepatoma 7777.

Cytosolic fractions B (salted out between 51-70% ammonium sulphate saturation) from rat liver and Morris hepatoma 7777, containing pyruvate kinase (EC 2.7.1.40) M2 isoenzymes, were purified by affinity chromatography on Blue Sepharose CL-6B. When compared by polyacrylamide gel electrophoresis at pH 8.3, all three M2 pyruvate kinase variants from Morris hepatoma 7777 had lower mobilities (alpha2, beta2, gamma3) than the three corresponding variants (alpha1, beta1, gamma2) from normal rat liver. Using an automatic amino-acid analyser, significant differences in selected amino-acid content have been found in corresponding highly purified gamma3 and gamma2 variants from Morris hepatoma and normal rat liver, respectively. The gamma3-variant of the Morris hepatoma M2 isoenzyme had twice the amount of L-tyrosine and L-cysteine, and a content of L-serine higher by 20% than the corresponding gamma2 variant of the normal rat liver M2 isoenzyme. It contained, however, significantly less dicarboxylic amino acids which explains its lower electrophoretic mobility. It showed also a decrease (by about 10%) in several other amino-acid content, corresponding to a 10% decrease in the tumour enzyme molecular mass.

Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution.

Lipoxygenases, which are widely distributed among plant and animal species, are Fe-containing dioxygenases that act on lipids containing (Z,Z)-pentadiene moieties in the synthesis of compounds with a variety of functions. Utilizing an improved strategy of data collection, low temperature, and synchrotron radiation of short wavelength, the structure of ferrous soybean lipoxygenase L-1, a single chain protein of 839 amino acid residues, has been determined by X-ray crystallography to a resolution of 1.4 A. The R-factor for the refined model is 19.7%. General features of the protein structure were found to be consistent with the results of prior crystallographic studies at lower (2.6 A) resolution. In contrast to the prior studies, the binding of a water molecule to the active site Fe was established. The octahedral coordination sphere of the Fe also includes the side chains of His499, His504, His690, and Asn694 as well as the terminal carboxylate of Ile839, which binds as a monodentate ligand. Asn694 is involved in a number of labile polar interactions with other protein groups, including an amide-aromatic hydrogen bond, and appears to be a weak ligand. Several possible access routes for dioxygen and fatty acids to the internal active site and substrate binding cavity are described. The protein structure restricts access to the Fe site such that the formation of an organo-Fe intermediate seems improbable. Structural restrictions pertinent to other proposed reaction intermediates, such as planar pentadienyl and nonplanar allyl radicals, are also discussed.

Crystallization and preliminary X-ray investigation of lipoxygenase-3 from soybeans.

Soybean lipoxygenase-3 has been crystallized by the vapor diffusion method in 16-20% polyethylene glycol (average M(r), 3,400), 0.2 M sodium acetate buffer, pH 5.7, at 21 degrees C, at a protein concentration of 8-15 mg/mL. The crystals, which diffract to 3-A spacings, belong to the monoclinic space group C2. Cell constants are a = 111.9, b = 136.4, and c = 61.6 A and beta = 95.7 degrees. The calculated value of Matthews's constant, Vm = 2.48 A3/kDa, is consistent with the presence of one molecule of lipoxygenase per crystallographic asymmetric unit (Z = 4).

Crystallographic determination of the active site iron and its ligands in soybean lipoxygenase L-1.

Five ligands of the active site iron atom in soybean lipoxygenase L-1 have been identified from the electron density map of the crystallized enzyme. The position of the iron atom can be readily and independently located from an anomalous difference electron density map. The ligands identified are His-499, His-504, His-690, Asn-694, and Ile-839, the carboxy-terminal residue. Our previous view that these three histidines are essential for activity and binding of iron, based on site-specific mutation studies, is confirmed. A sixth protein ligand is not present, and the sixth coordination site opens into a wide cleft. The structure of the soybean lipoxygenase was solved by multiple anomalous isomorphous replacements.

Identification of the iron-binding histidine residues in soybean lipoxygenase L-1.

Lipoxygenases constitute a class of non-heme, non-sulfur iron dioxygenases acting upon lipids possessing a 1,4-cis-cis-pentadiene moiety. The iron is known to be essential for activity. A motif of six histidine residues has been found in all of the thirteen lipoxygenases, from both plant and animal sources, whose sequences have been reported. We had previously obtained mutant proteins in which each of the 6 conserved histidines of soybean lipoxygenase L-1 had been replaced and found that the mutants H499Q, H504Q (or H504S) and H690Q had no detectable enzymatic activity. We have now found that these inactive proteins contain no Fe, although they have the same electrophoretic mobility as wild-type L-1 under both denaturing and non-denaturing conditions and react with anti-L-1 antibodies.

Conserved histidine residues in soybean lipoxygenase: functional consequences of their replacement.

Sequences of 13 lipoxygenases from various plant and mammalian species, thus far reported, display a motif of 38 amino acid residues which includes 5 conserved histidines and a 6th histidine about 160 residues downstream. These residues occur at positions 494, 499, 504, 522, 531, and 690 in soybean lipoxygenase isozyme L-1. Since the participation of iron in the lipoxygenase reaction has been established and existing evidence based on Mössbauer and EXAFS spectroscopy suggests that histidines may be involved in iron binding, the effect of the above residues has been examined in soybean lipoxygenase L-1. Six singly mutated lipoxygenases have been produced in which each of the His residues has been replaced with glutamine. Two additional mutants have been constructed wherein the codons for His-494 and His-504 have been replaced by serine codons. All of the mutant lipoxygenases, which were obtained by expression in Escherichia coli, have mobilities identical to that of the wild-type enzyme on denaturing gel electrophoresis and respond to lipoxygenase antibodies. The mutated proteins H499Q, H504Q, H504S, and H690Q are virtually inactive, while H522Q has about 1% of the wild-type activity. H494Q, H494S, and H531Q are about 37%, 8%, and 20% as active as the wild type, respectively. His-517 is conserved in the several lipoxygenase isozymes but not in the animal isozymes. The mutant H517Q has about 33% of the wild-type activity. The inactive mutants, H499Q, H504Q, H504S, and H690Q, become insoluble when heated for 3 min at 65 degrees C, as does H522Q. The other mutants and the wild-type are stable under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli.

We have constructed a full-length cDNA that encodes soybean seed lipoxygenase L-1 and have expressed it in Escherichia coli. This gene was inserted into a pT7-7 expression vector, containing the T7 RNA polymerase promoter. E. coli, strain BL21 (DE3), which carries the T7 promoter in its genome, was transfected with the plasmid. Expression of this gene when the cells were cultured at 37 degrees C yielded polypeptide that was recognized by anti-L-1 antibody, but had very little lipoxygenase activity. Yields of active enzyme were markedly increased when cells were cultured at 15-20 degrees C. When ethanol, which has been reported to be an excellent elicitor of heat-shock proteins in E. coli, was also present at a level of 3% the yield was further increased by 40%. Under optimum conditions 22-30 mg of soluble active enzyme was obtained per liter of culture.

Crystallization and preliminary X-ray investigation of lipoxygenase 1 from soybeans.

Soybean lipoxygenase 1 has been crystallized by the vapor diffusion method in 8-10% polyethylene glycol (average Mr 3400), 0.2 M sodium acetate buffer, pH 5.2-5.6, at a protein concentration of 6-12 mg/ml. Microseeding was employed to obtain growth of large single crystals. The crystals, which diffract to at least 2.2-A spacings, are monoclinic and of space group P2(1). Cell constants are: a = 95.4, b = 94.2, and c = 50.4 A and beta = 91.4 degrees. The calculated value of Vm (2.41 A3/Da) is consistent with the probable presence of one molecule of lipoxygenase/crystallographic asymmetric unit (Z = 2).

Crystallization of alcohol oxidase from Pichia pastoris. Secondary structure predictions indicate a domain with the eightfold beta/alpha-barrel fold.

Alcohol oxidase from Pichia pastoris has been crystallized from polyethylene glycol 4000 solutions. The crystals are tetragonal, a = 228 A, c = 456 A space group P4(1)2(1)2. The crystals scatter only to about 6 A resolution; their poor crystallinity may have some physiological function. Secondary structure predictions suggest that the C-terminal part of the molecule, residues 311-664, has the folding of an eightfold beta/alpha-barrel (TIM barrel). This would indicate common ancestry with four other flavoenzymes: canavalin, glycolate oxidase, flavocytochrome b, and trimethylamine dehydrogenase.

Primary structure of soybean lipoxygenase L-2.

The nucleotide sequence of soybean lipoxygenase-2 cDNA has been determined, and the complete amino acid sequence of the enzyme has been deduced. Limited direct amino acid sequence data for lipoxygenase-2 protein support this assignment and exclude mRNA representing lipoxygenase-1 and -3. Lipoxygenase 2 has a molecular weight of 97,036 and contains 865 amino acid residues, in contrast to the isozymes, lipoxygenase-1 and -3, which are known to contain 838 and 859 amino acid residues, respectively. Despite significant differences in behavior between these three isozymes, the amino acid sequences of lipoxygenase-1 and -3 are 81 and 74% identical to lipoxygenase-2, respectively. A region of 40 amino acid residues containing a cluster of six histidines and two tyrosines, which is highly conserved in all three isozymes, is discussed as a possible iron-binding region.

Primary structure of soybean lipoxygenase-1.

The primary structure of lipoxygenase-1 from soybeans has been determined. The results were deduced from the nucleotide sequence of the complete cDNA for isozyme lipoxygenase-1 and were confirmed by Edman sequencing of selected peptides obtained by CNBr cleavage and supported by carboxyl-terminal sequencing of the intact protein and by partial peptide analysis employing fast atom bombardment-mass spectroscopy. The enzyme has a molecular weight of 94,038 based on its content of 838 amino acids.

Catalytic and immunochemical properties of arylsulphatase A from urine, modified by potassium ferrate.

Arylsulphatase A (EC 3.1.6.1.) from urine was inactivated with potassium ferrate, a strong oxidizing agent. The inhibition could be prevented by competitive inhibitors, tetraborate and orthophosphate. Tetraborate which was shown to be a powerful competitive inhibitor (determined Ki = 4 X 10(-5) M) gave more efficient protection. The partially inactivated enzyme exhibited a Km value similar to that of the unmodified arylsulphatase A, and its Vmax decreased in proportion to the loss of enzymatic activity. The partially modified enzyme did not lose its ability to catalyse hydrolysis of p-nitrocatechol sulphate according to the "anomalous kinetics" exhibited towards this substrate and characteristic for arylsulphatase A. The immunochemical properties of arylsulphatase A either fully or partially inactivated were similar to those of the native enzyme. The results allow to conclude that ferrate reacts with arylsulphatase A in its active site. Thus ferrate seems to be a very sensitive probe for amino acid residues essential for catalytic activity of arylsulphatase A.