Rudolf Ehwald

Recording of subcutaneous glucose dynamics by a viscometric affinity sensor

Aims/hypothesis. To provide a nonenzymatic sensor for glucose monitoring in subcutaneous tissue. Methods. A continuously working affinity sensor based on the glucose-dependent viscosity of a sensitive liquid containing dextran and concanavalin A has been designed by arranging a microdialysis probe, two flow-resisting capillaries and two pressure transducers in a linear flow system. It allows synchronous processing of the viscosity of the sensitive liquid at the standard glucose concentration and the glucose concentration to be measured. In preliminary human trials the sensor was implanted into the subcutaneous tissue of the forearm and its read-out was compared with capillary blood concentrations. Results. In vitro, the viscometric sensor shows a linear and long-term stable dependence on the glucose concentration without detectable drift. At the applied flow rate of the sensitive liquid (about 5 μl/h) the technical delay is 5 to 10 min. The read-out of the implanted sensor followed the dynamics of the capillary blood glucose concentrations with a time-shift of 10 to 15 min but showed a systematic error when based on precalibration with polymer-free glucose solutions. After appropriate in vivo calibration, the read-out was in good or acceptable coincidence with capillary blood concentrations according to the error grid method and did not show any detectable reduction of sensitivity during the periods of measurement (up to 44 h). Conclusion/interpretion. The viscometric-affinity sensor is an efficient tool for current research on glucose monitoring in the subcutaneous tissue and can potentially be further developed for routine clinical use. [Diabetologia (2001) 44: 416–423]

Binding of Water, Oil, and Bile Acids to Dietary Fibers of the Cellan Type

Dietary fibers (DF) of the “cellan” type (consisting mainly or exclusively of undestroyed cells) were prepared as ethanol‐dried materials from apple, cabbage, sugar‐beet, soybean hulls, wheat bran, and suspension cultures of Chenopodium album L. and investigated with respect to their interactions with water, water−oil dispersions, bile acids, and oil. Water binding and retention capacities were found to be especially high in cellans obtained from thin‐walled raw material. Water damp sorption by dry cellans, when analyzed according to the GAB and BET equations, shows a considerable fraction of monolayer water. At a water activity of 0.98, the cell and capillary spaces outside the walls remained in the air‐filled state but the cell wall pores are filled with water. When the water content of a concentrated aqueous cellan suspension was equal to or below the water binding capacity, its rheological behavior was found to be of pseudoplastic nature. At a given dry weight concentration, yield stress and viscosity of such concentrated suspensions were highest for cellans with the highest water binding capacity. Dry cellan particles absorbed fatty oils without swelling but swell in a detergent‐stabilized oil/water emulsion with a similar liquid absorption capacity as in water. In contrast to the dry or alkane‐saturated cell wall, the hydrated wall is not permeable to oils in the absence of a detergent. Oil droplets may be entrapped within the cells, yielding a stable dispersion of oil in water. As DF of the cellan type absorb bile acids, preferentially glycoconjugates, from diluted solutions, they may have a potential to decrease the serum cholesterol level.

Cell wall microcapsules with different porosity from suspension cultured Chenopodium album

Abstract The primary cell wall of suspension cultured higher plant tissues may be used for size fractionation of macromolecules of different ranges of molecular mass by permeation chromatography, as the size limits of cell wall penetration may be adjusted by controlled decay of polygalacturonan. If care is taken to prevent pectin decay by β-elimination during the preparation of the vesicular packing material (VP) from suspension grown cell clusters of Chenopodium album (low temperature, pectin-preserving proteolysis buffer and washing solutions), extraction with ethanol and deproteinization with a pancreatic enzyme mixture did not significantly increase the size limit of permeation (SLP) of the native cell wall. However, the SLP was significantly increased after long term washing at room temperature or heat treatment with phosphate buffer at pH 7. If the VP was incubated in 2% Na 2 CO 3 solution at room temperature, its SLP increased significantly within the first 30 min but remained stable on further incubation in the alkaline solution. The extent of the porosity change of the cell wall occurring in Na 2 CO 3 solution may be varied by choice of the incubation temperature. Permeation chromatography using the variants of the VP with increased porosity enables separation between proteins with large peak separation in the range of middle (50–100 kD) and large (300 kD) molecular mass.

Determination of size limits of membrane separation in vesicle chromatography by fractionation of polydisperse dextran

Abstract A recently described vesicular chromatographic packing material (VP), consisting of purified plant cell walls with vesicular and cellular morphology, was characterized with respect to the minimum Stokes′ diameter necessary for complete exclusion (size limit of exclusion = SLE) and the maximum Stokes′ diameter permitting permeation into the whole stationary liquid volume (size limit of permeation = SLP). Using vesicle chromatography, the size fractionation of a polydisperse dextran preparation with a defined size distribution was carried out to determine the percentages of completely excluded ( P ex ) and completely permeable ( P perm ) dextran molecules. SLE may be derived from P ex and SLP from P perm taking into account the molecular size range of the fractionated polydisperse dextran sample. Values determined for the size limits of the vesicle membrane with the help of dextran 35 (calibrated on Sephadex G-200) were nearly equal to those determined with the help of dextran 15 (calibrated on Sephadex G-75). The SLP of the standard VP is 5.6 nm. However, negatively charged proteins with a Stokes′ diameter slightly below the SLP (pepsin and ovalbumin) are excluded from the VP. The method may be applied in controlling the separation limits of VPs with altered ultrafiltration properties.

Compensation of Temperature and Concanavalin A Concentratration Effects for Glucose Determination by the Viscometric Affinity Assay

A viscometer suitable for rapid measurements in small volumes of highly viscous liquids is described. Using this device the viscometric affinity assay for glucose was studied under variable conditions in order to obtain basic information for the design of a viscometric glucose sensor. The viscosity of the dextran/Concanavalin A (ConA) solution is sensitive to glucose in a broad range of the shear stress. However, for measuring the glucose concentration with this sensitive liquid the strong dependence of the absolute viscosity on temperature and ConA concentration has to be taken into account. For the purpose of calibration a parameter more suitable than the absolute viscosity is the relative fluidity (Fr) that is defined by the actual measured viscosity at a given glucose concentration, the reference viscosity at a standard glucose concentration, and a constant linearization coefficient. Fr shows a linear dependence on the glucose concentration in the therapeutically interesting range up to 30 mM and is not significantly changed by moderate variations of the ConA concentration or temperature.

A BATCH CULTURE METHOD FOR MICROALGAE AND CYANOBACTERIA WITH CO2 SUPPLY THROUGH POLYETHYLENE MEMBRANES1

A new method for CO2 supply to photoautotrophic organisms was developed, and its applicability for measuring specific growth rates in shaken batch cultures of cyanobacteria and unicellular algae was shown. Small bags containing a concentrated carbonate buffer with a CO2 partial pressure of 32 mbar were prepared from a thin foil of low density polyethylene (LDPE). These bags were inserted as CO2 reservoirs (CRs) into polystyrene culture flasks with gas‐permeable screw caps, which were suitable to photometric growth measurement. CO2 was released directly into the medium with membrane‐controlled kinetics. The CRs were not depleted within 1 week, although the atmosphere in the culture vessel exchanged rapidly with the ambient air. Rates of initial growth and final densities of the cultures of six different unicellular algal species and one cyanobacterium were markedly increased by diffusive CO2 supply from the CR. In the presence of a CR, growth was exponential during the first 2 d in all cultures studied. The method described allowed a high number of measurements of specific growth rates with relatively simple experimental setup.

Analysis and purification of monomethoxy-polyethylene glycol by vesicle and gel permeation chromatography

Abstract Vesicle chromatography (VC) and gel permeation chromatography (GPC) were used for characterisation and purification of monomethoxy-polyethylene glycol (M-PEG), a reagent for protein modification. Detection of low concentrations of contaminating PEG was facilitated by a very sensitive colourimetric detection method with a detection limit of 1 μg/ml. For analytical purposes GPC on Superose 12 was superior to VC. Molecular masses, polydispersity and percentage of contaminating PEG were estimated. As a comparison 1H NMR spectroscopy was carried out. The results were in good accordance with GPC. A two-step preparative purification with VC of M-PEG containing 22.9% PEG reduced the PEG content to 4.4%.

A Special Role of the Rhizodermis in Nutrient Uptake by Plant Roots

Summary Using the possibility of selective killing the rhizodermis by means of osmotic shock further evidence is produced for the hypothesis of a restricted diffusion into the free space of a higher plants root cortex and of a special role of the rhizodermis in uptake processes of plant roots. Osmotic shock causes a strong decrease of phosphate uptake from low-concentrated solutions due to an osmotic rupture of rhizodermal cells. Phosphate uptake from higher concentrated solutions is only slightly influenced. It is concluded that phosphate uptake from low-concentrated solutions takes place at the shock-sensitive tissue surface only whereas higher concentrations permit the diffusion of the solute into the free space and, therefore, allow the shock-insensitive cortical cells to take part in the uptake process. This view is supported by a second line of evidence coming from the investigation of the time course and the temperature dependence of phosphate uptake and from measurements of the trans-membrane potential difference.

Post-Stress Thickening of Dextran/Concanavalin A Solutions Used as Sensitive Fluids in a Viscosimetric Affinity Assay for Glucose

Glucose‐dependent low‐stress and high‐stress rheology of a sensitive fluid (SF) containing high‐molecular‐weight dextran (30 g/L) and Concanavalin A (ConA, 10 g/L) was studied to define conditions for reproducible glucose determination by a viscosimetric sensor. The viscosity of the investigated SF was reproducibly glucose‐dependent only at sufficient shear stresses (>0.5 Pa) . Rheometric parameters measured at low stresses (<0.1 Pa) were strongly dependent on the shear history. Low‐stress viscosity measured after laminar flow with a high rate (90 s, 100 s−1, 4.5 Pa) increased by more than 2 orders of magnitude with slow kinetics. Solutions thickened after laminar flow acquired gel‐like properties (storage modulus nearly equal to loss modulus in a broad range of frequencies) . The highly viscous and gel‐like state developed after flow was stable up to a yield stress of ca. 0.05 Pa. The ConA‐mediated contribution to high‐stress viscosity was almost completely suppressed by the presence of glucose (100 mM), whereas thickening after flow was rather insensitive to the competing sugar. Interaction of diffusion‐controlled ligand exchange at the ConA binding sites with convective displacement of dextran molecules has been considered a main reason for the observed thickening effect. It is proposed that the shear field has an orientating effect on lectin distribution in the dextran matrix, whereby far‐reaching lamellar affinity clusters are developed. In the subsequently unloaded fluid, residues of these structures can reorientate and be cross‐linked to a weak metastable gel by redistributing ConA.

Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption

Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.