Effect of acetaldehyde and glutaraldehyde fixation on the surface properties of red blood cells as determined by partition in aqueous phases

A correlation between partition of cells in dextran/polyethylene glycol aqueous phase systems and their relative electrophoretic mobilities is often in evidence. Absence of such a correlation is indicative that partition measures surface properties other than charge at the plane of shear. Acetaldehyde- and glutaraldehyde-fixed erythrocytes were partitioned in two-polymer phases and their electrophoretic mobility examined in order to investigate the circumstances under which the above-indicated correlation holds. Aspects of the effect of fixation of cells on their surface properties was thereby obtained.

1. 1. Acetaldehyde-fixed and normal human red blood cells have similar partitions and similar electrophoretic mobilities; glutaraldehyde-fixed red cells display markedly increased partitions and mobilities.

2. 2. Lipid-extracted aldehyde-fixed cells have substantially increased partitions, but unchanged mobilities, when compared with the fixed cells from which they were prepared. This indicates that the removal of lipid exposes a higher density of charge groups deeper in the membrane than is measurable by electrophoresis under the conditions used.

3. 3. The countercurrent distribution obtained with fixed cells depends on the length of time chosen for phase settling. Short times result in a single distribution while longer settling times give rise to a two-peak distribution. The latter phenomenon probably arises from a time-dependent aggregation of the fixed cells in the phases.

4. 4. Electrophoretic examination of the glutaraldehyde-fixed cells from different cavities along the extraction train indicates that the fixation process does not eliminate differences in the relative electrophoretic mobilities of erythrocytes of different ages.

     

Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases.

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.     

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.     

Theoretical and practical aspects of glutaraldehyde fixation

Synopsis This review first considers the many structures put forward for glutaraldehyde, and the purification of the commercial material for chemical, histological and histochemical studies. Some practical and theoretical problems of tissue fixation with glutaraldehyde, including artefacts, are then discussed. The chemical reactions with amino acids and proteins are considered next together with the physical changes in the proteins during the reactions. The known reactions of glutaraldehyde with nucleic acids, lipids and mucosubstances are explored briefly.     

The partition of cis-parinaric acid and trans-parinaric acid among aqueous,fluid lipid,and solid lipid phases

Summary In this article, I review the current information concerning the partition of the fluorescent probes, cis-parinaric acid (9, 11, 13, 15-cis, trans, trans, cis-octadecatetraenoic acid) and trans-parinaric acid (9, 11, 13, 15-all trans-octadecatetraenoic acid) among aqueous, solid lipid, and fluid lipid phases. The association of these probes with lipid is described by a mole fraction partition coefficient whose value is typically in the range of 1–5 × 10⁶, a reasonable value in light of partition coefficients for other fatty acids between hydrophobic phases and water. The partition coefficient, in the absence of lipid phase changes, is relatively independent of temperature and only slightly dependent on the total aqueous probe concentration.In lipid samples which contain coexisting fluid and solid phases, trans-parinaric acid preferentially partitions into the solid phase, while cis-parinaric acid distributes nearly equally between fluid and solid phases. This partition behavior probably arises from the molecular shape of the cis and trans parinaric acid isomers. From measurements of the polarization of fluorescence of cis and trans parinaric acid in mixed lipid systems or membranes it is possible to evaluate the proportion of lipid components involved in phase changes or phase separation. From fluorescence energy transfer between protein typtophan residues and the parinaric acid isomers it is possible to gain information about the organization of lipids and proteins in membranes and model systems. I close the review by considering some of the membrane research areas where these probes and their various lipid derivatives may be particularly useful.     

Ultrasound-facilitated formalin fixation of biological specimens

Abstract

In a previous study, we showed that ultrasound can dramatically reduce the time required for tissue fixation in formalin. It generally is believed that ultrasound increases the speed of tissue fixation in two possible ways: 1) increasing the speed of penetration of fixative molecules into tissue samples and 2) increasing the speed of cross-linking reactions. We addressed here the second possible way by using protein solutions and cultured cells, which minimized the effects of the penetration factor. Proteins or cultured cells in solution were fixed with formalin with or without ultrasound irradiation. Fixed proteins and cell lysates then were separated by SDS-poly acrylamide gel electrophoresis and subjected to Western blotting to examine cross-linking formation in certain proteins. Unexpectedly, irradiation with ultrasound did not produce an observable difference in the rate of cross-linking in protein solutions. In similar experiments using cultured cells, however, we observed a significant reduction in recovery of certain proteins from cells fixed by formalin under the influence of ultrasound, which indicated that the ultrasound fixation procedure accelerated cross-linking formation within cells. Studies on protein and cell fixation without ultrasound showed that cross-linking formation was closely related to incubation temperature, which indicates that the heating function, which is inherently associated with ultrasound is another major factor in the ability of ultrasound to accelerate cross-linking.     

Transition of lipid phase in aqueous dispersions of diacylglyceryltrimethylhomoserine

The phase transition in the lipid phase of aqueous dispersions of diacylglyceryltrimethylhomoserine (DGTS) was measured by fluorescence depolarization of parinaric acid and differential scanning calorimetry. In both techniques, the phase transition temperatures (Tm) of 1-palmitoyl-2-stearoyl DGTS and of 1,2-distearoyl DGTS were 53 and 59 degrees C, respectively. Each of these Tm values was significantly higher than the Tm value of phosphatidylcholine with an identical combination of fatty acids. This suggests that the intermolecular interactions of DGTS molecules are slightly different from those of phosphatidylcholine molecules.     

Electromotive phenomena in partition of erythrocytes in aqueous polymer two phase systems

Measurement was made of the electrical potential between the two phases formed in an aqueous solution containing 5% dextran, 4% polyethylene glycol and varying concentrations of sodium chloride and sodium phosphate. Partition of the polycation DEAE-dextran-glycyltyrosine-¹²⁵I in such systems containing varying salt composition could be correlated with the measured electrical potential. Partion of human erythrocytes which have a negative surface charge was also correlated related with the measured electrical potential. Binding of DEAE-dextran-glycyltyrosine-¹²⁵I to human erythrocytes had less effect on their partitioning than might be expected from the number of positive charges bound to their surface.     

Interaction of N-alkylanthracyclines with lipid bilayers: correlations between partition coefficients, lipid phase distributions and thermotropic behavior

The thermotropic behavior of multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC), or of DPPC in admixture with cardiolipin or cholesterol, in the presence of various N-alkyl derivatives of both adriamycin and adriamycin-14-valerate has been investigated by high sensitivity differential scanning calorimetry. The analogues, particularly the 14-valerate derivatives, which were most lipophilic as judged by their lipid/buffer, and to a lesser extent by their octanol/buffer, partition coefficients, were the most effective in depressing the tm of the investigated lipids; correlations, however, were not absolute. Other factors, such as the distribution of the drugs between the solid and liquid-crystalline phases of the bilayer, were also important to the observed membrane perturbations. With all anthracyclines, however, no major changes in the transition enthalpy were observed. In the case of vesicles prepared from pure DPPC, curve fitting analysis based on ideal solution theory (J.M. Sturtevant (1984) Proc. Natl. Acad. Sci. USA 81, 1398-1400) applied at relatively low drug concentrations where single peak transitions were produced, adequately described the differential scanning calorimetric results. At high drug concentrations, however, the presence of multi-peak transitions were indicative of non-ideality.     

Determination of partition coefficient by the change of main phase transition

The molar partition coefficients of amphiphilic additives, e.g. local anesthetics, between the aqueous phase, the liquid crystal and the gel phase of lipid membrane can be determined based on a combination of phase transition data obtained at high and low concentrations of the lipid in aqueous phase. The data obtained at high lipid concentration allow to find the phase diagram lipid-additive in the aqueous environment. The combination of this diagram with data obtained at low lipid and additive concentrations provides direct information on the concentration of anesthetics in the lipid and thus allows the calculation of the partition coefficient.     

Perturbation of ribosome subunit interaction by glutaraldehyde fixation

Ultracentrifugal analysis of ribosomal purity is complicated by the rapid reequilibration of ribosomes with their subunits, and this is further enhanced by the effects of hydrostatic pressure. Fixation of the ribosome system prior to ultracentrifugal analysis supposedly freezes the reequilibration, and thus tends to obviate these difficulties. However, no redistribution of the ribosome-subunit population must be allowed to occur during fixation. Thus, it is necessary that fixation be extremely rapid compared to the ribosome-subunit reequilibration, in order to avoid errors in analysis. It was the purpose of this investigation to make a direct experimental comparison of the rates of these two processes, fixation and ribosome-subunit reequilibration, using the stopped-flow technique with a light-scattering detector, under a variety of buffer environment readjustments. The following findings resulted from this study: (1) Fixation in Tris buffers could not be followed by light scattering because of interaction of glutaraldehyde with Tris, leading to continuous production of high molecular weight contaminants. (2) Polymerization of glutaraldehyde leads to increased ultraviolet absorption, which must not be confused with scattering changes. (3) Undialyzed ribosome solutions prepared by dissolving stock suspensions stored at high levels of magnesium and univalent electrolyte into a known standard buffer produced solutions having free Mg2+ levels lower than those of original buffer, complicating kinetic observations and threatening ribosome stability. (4) Addition of malonic acid as a buffer for Mg2+ largely eliminated this problem. (5) The kinetics of glutaraldehyde were measured quantitatively by studying the perturbation of ribosome association and dissociation kinetics produced during shifts of Mg2+ levels. (6) At fixative levels not producing coagulation, fixation kinetics can be competitive with those of association-dissociation. (7) Mass action effects like those of dilution can be caused by fixation, and can give rise to excess subunits, which may be mistaken for loose couples in original ribosome preparations.     

The effect of the lipid composition on the partition of liposomes in aqueous two-phase systems

Liposomes have been partitioned in aqueous two-phase systems consisting of water, dextran, poly(ethylene glycol), salt and buffer. Liposomes were used as a model system in order to determine the contribution of the lipids on the partition of membrane particles. The liposomes were composed of phospholipids with different polar head groups and different degrees of unsaturation. The role of cholesterol was also investigated.The polar head group of the phospholipid plays a dominant role in determining the partition of liposomes, while the degree of unsaturation is of less importance, thereby indicating that partition in two-phase systems is a surface dependent method. Incorporation of cholesterol in liposomes reduces differences in partition between liposomes of various composition.