Strontium90 for determination of time since death.

Strontium90 (Sr90) is an artificial nuclear fission product of the atmospheric a-bomb testing between 1945 and 1979. It was spread throughout the atmosphere in the following years. Sr90 is an analogue to calcium and therefore enriched in human bones. Several studies especially in the 1960s and 1970s were undertaken to investigate the Sr90 burden and the resulting incorporated radiation in humans, but present studies are missing. In this study nine bone samples, three from 1931/32 and six from 1989 to 1994 were examined by measuring the Sr90 radiation. The samples from 1931/32 did not show any Sr90 activity. All the samples from 1989 and later showed a Sr90 activity, but the intensity was very variable. Subsequent investigations should be done to determine the cut-off year for measurable Sr90 activity. Furthermore the determination of a specific time since death depending on Sr90 activity should be possible, due to the ranging Sr90 pollution between 1950 and 1980 and different uptake in adolescents and adults.

Determination of intracellular conductivity from electrical breakdown measurements.

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 omega X cm for the intracellular resistivity of intact red blood cells is consistent with the literature.

Electro-fusion of cells: principles and potential for the future.

Exposure of cells or liposomes to a brief pulse of a strong electrical field can result in a reversible breakdown of the outer membrane. Such breakdown results in an increase in permeability of the plasmalemma, which however re-seals after a short incubation (i.e. the original impermeability is restored). Two or more cells in contact can be made to fuse by this process, provided that the contact is close enough and that the pulse of the electrical field is short enough not to damage the cells. Methods of achieving this contact by electrical and magnetic fields are described. The magnetic method does not demand the use of the low conductivity media used earlier. Other possible modifications of this flexible technique are also described, and used to show how the technique can be modified in future, and how it may be applied to the fields of membrane research, medicine and plant breeding.

Rotation of cells in an alternating electric field: the occurrence of a resonance frequency.

Cells suspended in a low-conducting medium were exposed to an alternating electric field whose frequency was altered between 1 kHz and 2 MHz. A resonance frequency was observed at which all suspended cells rotated about an axis normal to the field lines (when the electric field strength was larger than a threshold value of about 400 V/cm). This resonance frequency varied from species to species of cells (mesophyll protoplasts of Avena sativa = 20-40 kHz, human erythrocytes and ghost cells = 80-100 kHz, yeast cells = 140-180 kHz, Friend cells = 30-40 kHz, at room temperature). The resonance frequency of cell rotation was observed only under specific experimental conditions which excluded interference by reversible electrical breakdown of cell membranes and by gravitational forces. Glutardialdehyde fixed and heated cells exhibited no rotation in the frequency and field range investigated. The phenomenon of rotation is discussed in terms of dipole orientation within the membrane.

Electro-mechanical properties of human erythrocyte membranes: the pressure-dependence of potassium permeability.

Electrical breakdown of cell membranes is interpreted in terms of an electro-mechanical model. It postulates for certain finite membrane areas that the actual membrane thickness depends on the voltage across the membrane and the applied pressure. The magnitude of the membrane compression depends both on the dielectric constant and the compressive, elastic modulus transverse to the membrane plane. The theory predicts the existence of a critical absolute hydrostatic pressure at which the intrinsic membrane potential is sufficiently high to induce "mechanical" breakdown of the membrane. The theoretically expected value for the critical pressure depends on the assumption made both for the pressure-dependence of the elastic modulus of the membrane and of the intrinsic membrane potential. It is shown that the critical pressure is expected at about 65 M Pa. The prediction of a critical pressure could be verified by subjecting human erythrocytes to high pressures (up to 100 M Pa) in a hyperbaric chamber. The net potassium efflux in dependence on pressure was used as an criterion for breakdown. Whereas the potassium net efflux was linearly dependent on pressure up to 60 M Pa, a significant increase in potassium permeability was observed towards higher pressure in agreement with the theory. The increase in the net potassium efflux above 60 M Pa was reversible, as indicated by measurements in which the same erythrocyte sample was subjected to several consecutive pressure pulses. Temperature changes in the erythrocyte suspension during compression and decompression were so small (less than 2 degrees C) that they could not account for the observed effects.

Pressure-induced variations of K+-permeability as related to a possible reversible electrical breakdown in human erythrocytes.

Above a hydrostatic pressure of about 600 b a pronounced reversible increase in the net K+-efflux from human erythrocytes is observed. The effect is explained in terms of an electro-mechanical compression of the membrane, resulting in a reversible breakdown of the membrane.

Immobilization of human red blood cells.

Human red blood cells were immobilized in an alginate network which was cross-linked with Ca2+ ions. The immobilized cells were stored for longer than 5 weeks at 4 degrees C in an isotonic buffered NaCl solution containing glucose, inosine, adenine, and guanosine for energy supply. The immobilized cells were released from the alginate network by dissolving the matrix with citrate. Both the immobilized and released cells retain their biconcave shape over the storage period. Measurements of the released cell population in a hydrodynamically focussing Coulter Counter demonstrated that the mean size of the size distribution, the breakdown voltage, and the internal conductivity have not changed in contrast to control measurements on red blood cells stored conventionally in suspension indicating that immobilization preserves cellular functions.

Erythrocytes and lymphocytes as drug carrier systems: techniques for entrapment of drugs in living cells.

Mouse thymocytes and erythrocytes are loaded electrically with drugs in isotonic solution. The loaded cells are used for targeting the drugs to specific sites in the organism in order to achieve a controlled drug release in time and space. The field technique used for the loading of the cells is based on the dielectric breakdown of the cell membrane which is observed when cell suspensions are subjected to external field pulses of 2-20 kV/cm for short time intervals (ns to microseconds). When an apparent membrane potential of about 1 V is reached in response to the external field, the membrane breaks down reversibly. The breakdown of the membrane is associated with a remarkable and reversible permeability increase of the cell membrane. The increase in permeability depends on the strength and the duration of the field pulse.

Electrical field effects induced in membranes of developing chloroplasts.

Etioplasts, etiochloroplasts, and chloroplasts of Avena sativa L. purified on a Percoll gradient were subjected to increasing electric field strengths in the orifice of a hydrodynamically focussing Coulter Counter. The change in resistance of the orifice when an organelle is present correlates well with the size of the plastid for field strengths up to about 3.5 kV cm(-1). Beyond this field strength, depending on the size of the organelle, the size is underestimated. The underestimation of the size is caused by the dielectric breakdown of the envelope membranes once a critical membrane potential has been exceeded. Beyond breakdown the signal of the particle is predominately determined both by the internal conductivity and the increased membrane conductivity. Measurements of the breakdown voltage of different developmental stages of the plastids reveal that the breakdown voltage decreases from 1.2 V in etioplasts to about 0.9 V in chloroplasts after 48 h illumination. The decrease in breakdown voltage can be explained in terms of increasing incorporation of proteins into the inner envelope membrane during development.This view is consistent with conclusions drawn by other authors from transport and biochemical studies. The underestimation of the size beyond breakdown is about 20% and increases to a constant value of about 40% during the first 3 h of illumination. The underestimation decreases again to about 10% when the chloroplast stage is reached. This result is consistent with the current view of chloroplast development. Mobilisation of glucans, the transformation of the prolamellar body of etioplasts into thylacoid membranes as well as an intensive synthesis of pigments and enhanced rates of ions transport in the first hour of illumination gives rise to an increased pool of ionic compounds within the plastid stroma.It should be noted that purification of the plastids on Percoll gradient leads to size distributions which are almost normally distributed over the whole field range, suggesting that the preparations are also electrically homogeneous (U. Zimmermann, F. Riemann and G. Pilwat: Biochim. Biophys. Acta 436, 460-474 (1976)). In contrast with results of Lürssen, K., Z. Naturforsch. 25b, 1113-1119 (1970) only a slight increase of the modal volume from the etioplast stage to the chloroplast stage is observed.

Electrical hemolysis of human and bovine red blood cells.

The external electric field strength required for electrical hemolysis of human red blood cells depends sensitively on the composition of the external medium. In isotonic NaCl und KCl solutions the onset of electrical hemolysis is observed at 4 kV per cm and 50 per cent hemolysis at 6 kV per cm, whereas increasing concentrations of phosphate, sulphate, sucrose, inulin and EDTA shift the onset and the 50 per cent hemolysis-value to higher field strengths. The most pronounced effect is observed for inulin and EDTA. In the presence of these substances the threshold value of the electric field strength is shifted to 14 kV per cm. This is in contrast to the dielectric breakdown voltage of human red blood cells which is unaltered by these substances and was measured to be approximately 1 V corresponding in the electrolytical discharge chamber to an external electric field strength of 2 to 3 kV per cm. On the other hand, dielectric breakdown of bovine red blood cell membranes occurs in NaCl solution at 4 to 5 kV per cm and is coupled directly with hemoglobin release. The electrical hemolysis of cells of this species is unaffected by the above substances with exception of inulin. Inulin suppressed the electrical hemolysis up to 15 kV per cm. The data can be explained by the assumption that the reflection coefficients of the membranes of these two species to bivalent anions and uncharged molecules are field-dependent to a different extent. This explanation implies that electrical hemolysis is a secondary process of osmotic nature induced by the reversible permeability change of the membrane (dielectric breakdown) in response to an electric field. This view is supported by the observation that the mean volumes of ghost cells obtained by electrical hemolysis can be changed by changing the external phosphate concentration during hemolysis and resealing, or by subjecting the cells to a transient osmotic stress immediately after the electrical hemolysis step. An interesting finding is that the breakdown voltage, although constant throughout each normally distributed ghost size distribution, increases with increasing mean volume of the ghost populations.

[Organ specific application of drugs by means of cellular capsule systems (author's transl)]. / Organspezifische Applikation von pharmazeutisch aktiven Substanzen über zellulare Trägersysteme

It is suggested to use living cells (red blood cells, lymphocytes and leucocytes) as drug delivery systems for temporal and spatial drug administration in human therapeutics and diagnosis. The effectiveness of drug loaded cells is demonstrated for the drug methotrexate which is used in cancer treatment. Red blood cells are loaded with methotrexate using the dielectric breakdown technique. Dielectric breakdown leads to a transient increase of permeability of the cell membrane. Red blood cells loaded with tritium-labelled methotrexate were injected into mice and the activity level was measured in several organs as a function of time. It is shown that with this drug delivery system more than 50% of the drug (after 10 min) can be accumulated in the liver and that a high activity level can be sustained in this or gan for more than 3 hours. On the other hand, administration of this drug by injecting solutions in the usual manner leads only to an 25% accumulation of methotrexate (after 10 min) in the liver. The drug is excreted completely after 1 to 2 hours. It is proposed to load red blood cells simultaneously with para- or ferromagnetic substances to obtain organ-specificity for any selected site of the body.

Enzyme loading of electrically homogeneous human red blood cell ghosts prepared by dielelctric breakdown.

Human red blood cell ghosts were prepared by electrical haemolysis at 0 degrees C in isotonic solutions using a discharge chamber which was part of a high voltage circuit. The size distribution of the ghosts was normally distributed, the modal (=mean) volume was approx. 115 mum3, performing the electrical haemolysis in the following solution: 105 mM KCI, 20 mM NaCL, 4mM MgCl2, 7.6 mM Na2HPO4, 2.94 mM NaH2PO4, 10 mM glucose, pH 7.2. Resealing was carried out at o degrees C for 10 min (after the haemolytic step) and then for further 20 min at 37 degrees C. The mean volume of the ghost preparation could be changed by variation of the phosphate concentration in the above solution replacing a part of NaCl by phosphate (5 mM phosphate: 94 mum3, 15 mM phosphate: 135 mum3). The breakdown voltage of the ghost cell membranes measured with a hydrodynamic focusing Coulter Counter depends on the mean volume (94 mum3 = 1.04 V, 134 mum3 = 1.36 V). On the other hand, the breakdown voltage is constant throughout each size distribution pointing to an "electrically homogeneous" ghost preparation. The sensitiviity of the Coulter Counter to detect electrical inhomogeneities in the membranes of a ghost population is demonstrated by dielectric breakdown measurements of an apparently normally distributed ghost preparation containing two different "electrically homogeneous" ghost population i.e. with two different breakdown voltages. The ghost cells obtained by electrical haemolysis in the above solution containing 10mM phosphate were fairly impermeable to sucrose and behave like an ideal osometer. It is further demonstrated that ghost cells can be loaded with enzymes (e.g. urease) and drugs using this technique and that these loaded ghost cells can be used as bioactive capsules for clinical application.

Dielectric breakdown measurements of human and bovine erythrocyte membranes using benzyl alcohol as a probe molecule.

Dielectric breakdown of intact erythrocytes and subsequent haemolysis in the presence of increasing concentrations of benzyl alcohol were investigated by means of an electrolytical discharge chamber and a hydrodynamic focusing Coulter Counter. Low concentrations of the drug stabilized human and bovine erythrocytes against haemolysis induced by dielectric breakdown of the cell membrane in isotonic solutions, while high concentrations caused lysis similar to hypotonic and mechanical haemolysis. The stabilizing effect of the drug on electrically induced haemolysis depends on the pulse length of the applied electric field. The critical dielectric breakdown voltage of the membranes of intact cells decreases progressively with increasing benzyl alcohol concentrations, at which the membrane is also more stabilized against electrical and osmotic haemolysis. Occasionally, an increase in the dielectric breakdown voltage is observed at drug concentrations at which lysis occurs. A similar depedence of the breakdown voltage on drug concentration was found for human erythrocyte ghost cells prepared by dielectric breakdown. The results are consistent with the electromechanical model suggested for the dielectric breakdwon mechanism and with the assumption of Metcalfe, using NMR and ESR techniques, that the fluidity of the membrane increases with increasing benzyl alcohol concentration.

Release and uptake of haemoglobin and ions in red blood cells induced by dielectric breakdown.

External electric field strengths of the order of 10-3 minus10-4 v-cm-minus1 induce potassium release and concomitant sodium uptake in human and bovine red blood cells, as demonstrated in an electrolytic discharge chamber. The reversible increase of the membrane permeability once the critical membrane potential is reached is caused by dielectric breakdown of the membrane. The values of the critical membrane potential differences calculated from the potassium release and sodium uptake curves are close to those which were calculated from dielectric breakdown measurements in a hydrodynamic focussing Coulter Counter using the Laplace equation. With bovine red blood cells, the potassium release and the concomitant sodium uptake is coupled with haemoglobin release from the cells, while with human red blood cells much higher external electric field strengths are required for haemoglobin release. The external electric field strength required for solute release and uptake in bovine and human red blood cells depends on the pulse length, particularly below a value of about 10 mus, when a strong increase in the field strength occurs with decreasing pulse lengths. At 50-100 mus pulse lengths an asymptotic value of the critical electrical field strength of 2.6 kV-cm-minus1 for the modal volume of human red blood cells and 2.8 kV-cm-minus1 for the modal volume of bovine red blood cells is reached, corresponding to a critical membrane potential difference of about 1.1 V for both species. This value is close to that measured directly for dielectric breakdown of the membranes of Valonia utricularis (0.85 V, 20 degrees C). The increase in electric field strength with decreasing pulse length can be explained by the capacitance of the membrane, which becomes the rate limiting step for the temporal build-up of the electric potential across the membrane. The time constant of this process was determined to be approx. 10 mus. The critical membrane potential difference for breakdown is therefore pulse-length independent. The breakdown of the membrane can be interpreted by an electromechanical collapse of the membrane material. Numerical considerations of the dynamics of this membrane collapse predict that the breakdown time is a very rapid process.