K. Schmidt

Lipid transfer proteins.

Translocations of various lipid species between membranes have been extensively studied. The transport of water-insoluble lipids is thought to require the participation of lipid transfer proteins (LTP). Several LTP, differing in their physiochemical properties and substrate specificities, have been purified to homogeneity from blood plasma, eucaryotic and procaryotic cells. Depending on their site of activity, they can be classified as extracellular and intracellular LTP. Extracellular LTP are found in the blood plasma and intracellular LTP, which were originally characterized as phospholipid exchange proteins, are ubiquitous in nature. Despite the enormous knowledge about their physicochemical properties and their function in vitro their physiological role has not been clearly demonstrated. However, their ubiquitous occurrence indicates an important role in cellular events. This review gives an overview of this interesting category of proteins, which are able to catalyze inter-membrane transfer and exchange of lipids.

Prolongation of Corneal Allograft Survival with Liposome-encapsulated Cyclosporine in the Rat Eye

PURPOSE To study the effects of different formulations of topical cyclosporine (Cyclosporin A [CsA]) on corneal allograft rejection in a rat model. METHODS Female Lewis rats received penetrating keratoplasties from female Wistar-Furth donors. A total of 78 allogeneic grafts were performed. An additional 15 syngeneic grafts (Lewis) were used as technical controls. Two CsA preparations with equivalent drug concentrations (2.1 mg/ml) were applied as drops: CsA encapsulated in large unilamellar liposomes (CsA-LIP) and CsA dissolved in olive oil (CsA-DR). Allogeneic grafts were randomly assigned to receive CsA-LIP or CsA-DR beginning on the day of surgery five times daily for 10 days. Animals without any treatment or receiving empty liposomes (EM-LIP) were used as treatment controls. Grafts were graded three times weekly and a rejection index was generated based on graft clarity, neovascularization, and vessel size. RESULTS All syngeneic grafts remained clear over the observation period of 60 days. Rejected allogeneic grafts without any treatment and those receiving EM-LIP or CsA-DR showed a mean survival time (+/- standard deviation) of 14 +/- 4, 14 +/- 5, and 14 +/- 4 days, respectively. There was no significant difference in mean survival time between the grafts without any treatment and those in CsA-DR or EM-LIP treatment groups. The mean survival time of rejected grafts in animals receiving CsA-LIP was prolonged to 20 +/- 4 days. There was a significant difference in the mean survival time between the CsA-LIP treatment group and groups receiving CsA-DR, EM-LIP, or no treatment (P < or = 0.05). The Kaplan-Meier survival curve of the CsA-LIP treatment group was significantly different from the other experimental groups. The graft survival rate in the CsA-LIP group was 77%, whereas the rate was 37% in the non-treated group, 45% in the CsA-DR group, and 36% in the EM-LIP group. CONCLUSION Encapsulation of CsA in liposomes might be a promising formulation for use in the prevention of corneal graft rejection.

Studies on the mechanism of bile salt-induced liposomal membrane damage.

The damage of phosphatidylcholine membranes by bile salts such as cholate, deoxycholate (DC), chenodeoxycholate (CDC), ursodeoxycholate (UDC), as well as their glyco- and tauroconjugates, and lithocholate (LC) were studied. The permeabilities of liposomes differing in size (700 and 1,700 A in diameter) were determined at increasing bile salt concentrations. The release of entrapped raffinose (3H) (MW: 594) or inulin (3H) (MW: 5,000) was measured by pelleting of the liposomes and subsequent determination of the radioactivities in the supernatant. The release of these uncharged volume markers of different size points to a formation of membrane leaks increasing in size with increasing bile salt concentration. Determination of the membrane damaging threshold concentrations of bile salts demonstrated a higher stability of the smaller liposomes. Incubation of the smaller liposomes with increasing DC concentrations results in a successive substitution of lecithin by DC. The predominantly DC-containing vesicles are of remarkable stability against higher DC concentrations. The damaging properties of bile salts increase with decreasing number of hydroxy groups, with the exception of UDC and its conjugates which are much less membrane toxic than the other dihydroxy bile salts. Conjugation with glycine or taurine slightly enhances the membrane toxicity of bile salts with the exception of UDC. Sulfation of the 3-alpha-hydroxy group of LC reduces the damaging effect to about 10%.

Loading of preformed liposomes with high trapping efficiency by detergent-induced formation of transient membrane holes

Abstract Transient membrane holes induced by sodium cholate (Schubert et al., 1986, Biochemistry 25, 5263–5269) were used to encapsulate molecules at least up to M r = 70,000 into preformed liposomes. High trapping efficiencies (TE) close to 50% were achieved with concentrated suspensions of liposomes independent of their lamellarity, which was evaluated by cryomicroscopy of cryofixated suspensions. Another suitable hole-forming detergent is n -octyl β- d -glucopyranoside. Maximum trapping efficiency was measured within a narrow concentration range of added detergent below the onset of membrane solubilization. Using pure egg yolk lecithin (egg PC) or egg PC/bovine brain phosphatidylserine (7:3 mol/mol), trapping efficiency compared with the ratio of the volume inside the liposomes to the total aqueous volume in the samples. Therefore, it is probable that solutes that have been added to the external liposomal space diffuse into the liposomes, when the membranes are opened for several minutes. TE is slightly reduced by 30 mol% cholesterol in the membrane or if highly concentrated protein solutions (insulin or hemoglobin) or molecules with elongated form (polyuridylic acid) are to be encapsulated. An especially low TE into DPPC liposomes at 25°C is indicative of suppressed hole formation in the gel phase of the membrane. The residual amount of detergent after rechromatography of the loaded liposomes and liposome alterations are negligible. Detergent-induced liposome loading (DILL) is a convenient method for entrapping small molecules, larger carbohydrates, proteins, and nucleic acids into preformed liposomes with membranes in the liquid crystal phase. In addition, the ratio of calculated to found trapping efficiency of inulin is a suitable value for the mean lamellarity of vesicles up to a diameter of 200 nm.

Preparation of hemoglobin-containing liposomes using octyl glucoside and octyltetraoxyethylene

Hemoglobin (Hb) was encapsulated in phosphatidylcholine vesicles by removal of the detergent n-octyl beta-D-glucoside (OG) or n-octyltetraoxyethylene (C8E4) out of mixed detergent-lipid micelles in Hb solution. Three types of apparatus were used for dialysis. Dialysis buffer flow rates, the surface area of the dialysis membranes, and detergent-lipid interactions determined the rate of dialysis, which influenced liposome size and lamellarity. Slow dialysis led to the formation of multilamellar liposomes, at increased dialysis rates Hb liposomes became smaller and unilamellar. Hb was enclosed at highest concentrations in larger liposomes, which included the negatively charged lipid phosphatidylserine or phosphatidic acid as a membrane component. Co-encapsulation of the allosteric factor inositol hexaphosphate led to oxygen dissociation curve values almost identical to those of whole blood. The oxygen-release capacity of Hb liposome suspensions in the physiological partial pressure range was comparable to whole blood. Storage of Hb liposomes for 2 months leaves oxygen-carrying characteristics virtually unchanged, with met-Hb levels increasing to only 11% of total Hb. Preparation of Hb liposomes by dialysis of octyl glucoside or C8E4 is a mild and efficient method for encapsulation of Hb. Since these Hb liposomes can be produced in scale-up batch sizes, they are a candidate for use as an oxygen-carrying blood surrogate.

A model to reproduce predictable full-thickness burns in an experimental 0animal

A standardized, reproducible animal model is a prerequisite to study concepts in the therapy of extensive burn injuries. The development of a new model makes it possible to produce predetermined burn injuries with a set temperature, time, contact pressure, and standard extent of tissue damage. For our studies we chose rats and exposed them to a temperature of 250 degrees C for 20 s and a contact pressure of 500 g/cm2 over various percentages of TBSA (total body surface area). The animals received shock prophylaxis for 3 days postburn and were kept under standardized conditions in a laminar airflow compartment. The temperature was kept at 32 degrees C and the relative humidity at 75 per cent. To reduce bacterial contamination, air was filtered through special bacteria-proof filters. Under these conditions we found burns of approximately 35 per cent TBSA to be sublethal resulting in 80 per cent mortality between days 5 and 7. This model permits the investigator to vary the burned skin area to any required extent for a reproducible study of different concepts of burn therapy.

New class of 5-lipoxygenase inhibitors: correlation between inhibition of LTB4 production and chemiluminescence of human polymorphonuclear granulocytes

The effect of several series of compounds on the biosynthesis of leukotriene B4 in human polymorphonuclear granulocytes (PMNL) was measured after stimulation of the cells by the Ca2+ ionophore A23187. Phenylhydrazone derivatives and some pyrrole derivatives strongly inhibited 5-lipoxygenase activity at 10 µM. In contrast, 4-aminopyrrolopyrimidines and 4-oxopyrrolopyrimidmes did not exhibit any effect. The compounds examined for inhibition of 5-lipoxygenase were also tested in a chemiluminescence assay for potential effects on the emission of photons as a measure of activated oxygen species generated by the stimulated granulocytes. There was a good correlation between lipoxygenase inhibition and suppression of chemiluminescence; however, some derivatives with no inhibitory activity against lipoxygenase still reduced chemiluminescence, which suggests that an alternative inhibitory mechanism of chemiluminescence must be present. Cyclooxygenase inhibitors such as aspirin did not suppress chemiluminescence of granulocytes. It is, therefore, unlikely that cyclooxygenase is required in the chemiluminescence reaction. In contrast, LTB4 seems to play an important role in the zymosan-induced chemiluminescence of PMNL.

Polyvinylpyrrolidon-gebundenes Hämoglobin als ein Sauerstoff übertragender Blutersatzstoff

SummaryThe development of blood substitute oxygen carriers has branched off into several directions: stroma-free hemoglobin solutions, modelling of hemoglobin by fixation of heme or hemopeptides to polymers, filling of microcapsules with hemoglobin solution, crosslinking of hemoglobin. However, the application as blood replacement fluids in patients has not been possible so far because of several disadvantages. In this paper synthesis and properties of an oxygen carrying blood substitute are described in which the hemoglobin molecule is covalently bound to polyvinylpyrrolidone. In aqueous solution the preparation fulfills some basic requirements such as high solubility, only slightly increased oxygen affinity, and low viscosity. Normothermic perfusion experiments with isolated pig kidneys demonstrate that the solution provides an adequate oxygen supply to the organ, has no toxic side effects on kidney function, and has a significantly lower elimination rate compared to an unmodified hemoglobin solution.ZusammenfassungDie Entwicklung Sauerstoff übertragender Blutersatzstoffe hat sich in mehrere Richtungen aufgespalten: stromafreie Hämoglobinlösung, Nachbildung von Hämoglobin durch Bindung von Häm bzw. Hämopeptiden an Polymere, Mikroverkapselung von Hämoglobinlösung, Quervernetzung von Hämoglobin. Dennoch war es bis heute nicht möglich, eine bei Patienten anwendbare Infusionslösung zu präparieren, da verschiedene Schwierigkeiten dies verhindern. In der vorliegenden Arbeit werden Synthese und Eigenschaften eines Sauerstoff übertragenden Blutersatzes beschrieben, bei dem das Hämoglobinmolekül kovalent an Polyvinylpyrrolidon gebunden ist.In wäßriger Lösung erfüllt das Syntheseprodukt einige grundlegende Erfordernisse wie z.B. hohe Löslichkeit, nur geringfügig gesteigerte Sauerstoffaffinität und niedrige Viskosität. Normotherme Perfusionsexperimente an isolierten Schweinenieren zeigen, daß die Lösung eine hinreichende Sauerstoffversorgung des Organs gewährleistet, keine toxischen Nebenwirkungen auf die Nierenfunktion hat und im Vergleich zu einer unmodifizierten Hämoglobinlösung wesentlich langsamer ausgeschieden wird.The development of blood substitute oxygen carriers has branched off into several directions: stroma-free hemoglobin solutions, modelling of hemoglobin by fixation of heme or hemopeptides to polymers, filling of microcapsules with hemoglobin solution, crosslinking of hemoglobin. However, the application as blood replacement fluids in patients has not been possible so far because of several disadvantages. In this paper synthesis and properties of an oxygen carrying blood substitute are described in which the hemoglobin molecule is covalently bound to polyvinylpyrrolidone. In aqueous solution the preparation fulfills some basic requirements such as high solubility, only slightly increased oxygen affinity, and low viscosity. Normothermic perfusion experiments with isolated pig kidneys demonstrate that the solution provides an adequate oxygen supply to the organ, has no toxic side effects on kidney function, and has a significantly lower elimination rate compared to an unmodified hemoglobin solution.

Selenium concentration and activity of glutathione peroxidase in lysate of human erythrocytes

ZusammenfassungDie Selenkonzentration in menschlichen Erythrozyten wurde nach nasser Veraschung des biologischen Materials mit Hilfe einer fluorometrischen Bestimmungsmethode gemessen. In den Erythrozyten derselben Blutproben wurde die Aktivität des Selenoenzyms Glutathionperoxydase (EC 1.11.1.9) mit tertiärem Butylhydroperoxyd als Akzeptorsubstrat bestimmt. Die Ergebnisse lassen erkennen, daß nur etwa 10% des gesamten Selengehaltes menschlicher Erythrozyten an das Enzym gebunden sind. Zwischen der Enzymaktivität und der Selenkonzentration besteht keine Korrelation. Auf der Grundlage dieser Ergebnisse kann vermutet werden, daß der protektive Effekt des Selens in Glutathionperoxydase gegen oxydative Zellschädigung nicht die einzige Funktion dieses Elements sein kann.SummaryThe concentration of selenium in human red cells was measured by a fluorometric determination method after wet digestion of the biological material. In the red cells from the same blood samples the activity of the selenoenzyme glutathione peroxidase (EC 1.11.1.9) was determined with tertiary butyl hydroperoxide as acceptor substrate. The results show that only 10% of the total selenium content of human red cells is fixed to the enzyme. No correlation between the enzyme activity and the selenium concentration could be found. On the basis of these results can be presumed that the protective effect of selenium in glutathione peroxidase against oxidant damage of cells is not the only biological function of this element.The concentration of selenium in human red cells was measured by a fluorometric determination method after wet digestion of the biological material. In the red cells from the same blood samples the activity of the selenoenzyem glutathione peroxidase (EC 1.11.1.9) was determined with tertiary butyl hydroperoxide as acceptor substrate. The results show that only 10% of the total selenium content of human red cells is fixed to the enzyme. No correlation between the enzyme activity and the selenium concentration could be found. On the basis of these results can be presumed that the protective effect of selenium in glutathione peroxidase against oxidant damage of cells is not the only biological function of this element.

Impregnation of collagen corneal shields with liposomes: Uptake and release of hydrophilic and lipophilic marker substances

PURPOSE Liposomes and collagen corneal shields (CCS) have been used as ophthalmic drug delivery devices. With regard to a possibly combined application, we studied the effects of surface charge and bilayer fluidity of liposomes on their uptake and release by CCS. METHODS 12-hours-CCS were soaked in large unilamellar liposomes, which had been labelled with 4,5-carboxyfluorescein (CF) and N-(lissamine rhodamine B sulfonyl)-diacyl-phosphatidylethanolamine (PE-RhB) in the aqueous space and in the liposome bilayer, respectively. Released fluorophores were determined fluorometrically in the elution buffer at intervals from 1 to 240 min after immersion. RESULTS The CF concentration in the CCS soaked in a CF solution was two to seven times higher than immersion in the liposome suspensions. Among those, the negatively charged, cholesterol-containing preparation led to the highest CF concentration in the CCS. The PE-RhB concentration was highest after soaking the CCS in neutral, cholesterol-free liposomes. All types of liposomes were found inside the CCS by freeze fracture electron microscopy. The release kinetics data indicate a first order release. More than 90% of CF was released by the CCS within the first 30 min. This was equal after soaking the CCS in the CF solution or in liposomes. With DOPC-liposomes, the maximal release was already attained after 10 min. In general, the differences in the release kinetics of both hydrophilic and lipophilic markers, obtained by the various liposome types were small. CONCLUSIONS Our results indicate that surface charge and bilayer fluidity are of minor importance for the interaction with collagen corneal shields. However, since the release kinetics of a liposome-encapsulated hydrophilic or lipophilic substance are similar to the release of a non-encapsulated drug, the combination of liposomes with collagen shields may be useful mainly with respect to the encapsulation of drugs which do not penetrate the ocular surface as well as to prolong corneal contact time of the liposomes.