Prediction of ice content in biological model solutions when frozen under high pressure

High pressure is, at least, as effective as cryoprotective agents (CPAs) and are used for decreasing both homogenous nucleation and freezing temperatures. This fact gives rise to a great variety of possible cryopreservation processes under high pressure. They have not been optimized yet, since they are relatively recent and are mainly based on the pressure–temperature phase diagram of pure water. Very few phase diagrams of biological material are available under pressure. This is owing to the lack of suitable equipment and to the difficulties encountered in carrying out the measurements. Different aqueous solutions of salt and CPAs as biological models are studied in the range of 0°C down to ‐35°C, 0.1 up to 250 MPa, and 0–20% w/w total solute concentration. The phase transition curves of glycerol and of sodium chloride with either glycerol or sucrose in aqueous solutions are determined in a high hydrostatic pressure vessel. The experimental phase diagrams of binary solutions were well described by a third‐degree polynomial equation. It was also shown that Robinson and Stokes' equation at high pressure succeeds in predicting the phase diagrams of both binary and ternary solutions. The solute cryoconcentration and the ice content were calculated as a function of temperature and pressure conditions during the freezing of a binary solution. This information should provide a basis upon which high‐pressure cryopreservation processes may be performed and the damages derived from ice formation evaluated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium--a revisit

We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to −50 °C or below occurs at temperatures where a critical fraction of the external water remains unfrozen [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53; P. Mazur, I.L. Pinn, F.W. Kleinhans, The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature, Cryobiology 54 (2007) 223-233]. For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. [E.J. Woods, M.A.J. Zieger, D.Y. Gao, J.K. Critser, Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/Water and their application to cryopreservation., Cryobiology 38 (1999) 403-407]. Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic [F.W. Kleinhans, P. Mazur, Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest, Cryobiology 54 (2007) 212-222]. Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. [J.F. Guenther, S. Seki, F.W. Kleinhans, K. Edashige, D.M. Roberts, P. Mazur, Extra-and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401-416] on IIF in oocytes of the frog Xenopus. It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol.     

Standard electromotive force of the H2-AgCl;Ag cell in 30, 40, and 50 mass% glycerol/water from -20 to 25 degrees C: pK2 and pH values for a standard "mops" buffer in 50 mass% glycerol/water

Data are presented regarding the establishment of the pH (designated pH*) of a standard buffer solution suitable as a pH reference in 50 mass% glycerol/water mixtures at temperatures ranging from -20 to 25 degrees C. The buffer material selected was the ampholyte Mops [(3-N-morpholino)-propane sulfonic acid], and the reference standard consists of equal molal amounts of Mops and its sodium salt. The assignment of pH* values is based on measurements of the electromotive force (emf) of cells without liquid junction of the type: Pt;H2(g, 1 atm) / Mops, Na Mopsate, NaCl / AgCl;Ag and the pH* was derived from a determination of K2, the equilibrium constant for the dissociation process (Mops) +/- in equilibrium with (Mopsate)- + H+. The standard emf of the silver-silver chloride electrode in 30, 40, and 50 mass% glycerol/water mixtures was determined from emf measurements of the cell at subzero temperatures with HCl solutions replacing the buffer-chloride mixtures.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C₁₈ solid-phase extraction column. After elution with 500 μl of elution solution, the penicillins are derivatized with 500 μl of 1,2,4-triazole—mercuric chloride solution at 65°C for 30 min. The penicillin—mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C₁₈ column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

Small-angle X-ray scattering and differential scanning calorimetry studies on reversibly modified human-serum low density lipoproteins.

Small-angle x-ray scattering diagrams of human serum low density lipoprotein (LDL) were recorded at several temperatures in solutions of different freezing points. It was found that modifications of the x-ray patterns observed on cooling the lipoprotein samples below 0 degrees C are due to reversible alterations of the LDL surface structure induced by the freezing process (independent of temperature). With both intact and partially dehydrated LDL, differential scanning calorimetry (DSC) carried out in the body temperature range revealed a heat absorption characteristic of the transition from a liquid crystal to an isotropic liquid phase of cholesteryl esters within the lipoproteins (Deckelbaum, R. J., Shipley, R. J., Small, P. M., Lees, R. S., & George, P. K. (1975) Science 190, 392). However, small-angle x-ray scattering diagrams recorded with the same LDL sample before and after the partial removal of water were found to be very different: the scattering curve for intact LDL showed a strong band centered at (36 A)-1 which disappeared upon drying and reappeared upon restoring the water. Our results suggest that the presence of this signal strongly depends on the molecular structure of the lipoprotein surface.     

Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA+salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.     

Liquid water-ice I phase diagrams under high pressure: sodium chloride and sucrose models for food systems

The knowledge of high pressure and low temperature phase diagrams of aqueous systems is required in fields such as food sciences, biology, cryo-microscopy and geology, to reduce processing costs, improve treatments results or advance in physical phenomena understanding. The phase transition curve between liquid water and ice I for sucrose and sodium chloride solutions has been obtained for concentrations ranging from 16% to 36% and from 1.63% to 16.09% (w/w), respectively. An accurate experimental method, based on the pressurization of an ice-solution mixture, adequate to build the entire phase transition curve at constant concentration, has been developed. Simon-like equations have been used to empirically describe the phase transition curves, so that they allow easy data interpolation.     

Phase-Separable Aqueous Amide Solutions as a Thermal History Indicator

Aqueous solutions of several new amide compounds for use as simple thermal history indicators in the low-temperature transport of food and other products were synthesized. The phase transition temperatures of the aqueous solutions can be freely adjusted by changing the amide-water ratio in solution, the sodium chloride concentration of the water, and the type of amide compound. It is expected that these aqueous solutions can be applied as new thermal history indicators.     

Studies of fluid exchanges between rat liver slices and simple media

1. The exchange of fluid between slices of rat liver and solutions of monosaccharides, disaccharides, and sodium chloride has been studied in relation to the temperature of incubation. The point of apparent isotonicity (P.A.I.) of the tissue was defined as the concentration of the solution in which the slices neither gained nor lost weight after immersion for a period of 10 minutes. 2. In solutions of glucose, the P.A.I. of the slices was significantly lower at 4°C. than at 20°C. but similar at 20°C. and 37°C. Upon immersion for 15 to 60 minutes in 0.66 molar glucose the slices always swelled more at 37°C. than at 20°C. In solutions of sucrose change in the temperature of incubation was without effect on the hydration of the tissues. 3. In solutions of sodium chloride, the P.A.I. and the content of water, chloride, and sodium plus potassium were lower at 37°C. than at 20°C. 4. These findings emphasize the role of translocation of solute in providing an osmotic gradient for the movement of water between the tissue slices and the media.     

Determination of biomass dry weight of marine microalgae

Total biomass dry weight (DW) and ash free dry weight (AFDW) of five species of marine microalgae, Dunaliella sp., Isochrysis galbana, Nannochloropsis sp., Nitzschiaclosterium and Porphyridium cruentum, retained on filter paper, were determined. Dunaliella and Isochrysis cells have no cell wall; Nannochloropsis, Nitzschia and Porphyridium possess a cell wall and Nitzschia and Porphyridium cells are covered by silica and mucilage coating, respectively. In all these algae, DW of non-washed samples was at least 1.2 times higher than those washed by distilled water,0.9% sodium chloride, 0.5 M ammonium formate or 0.5 M ammonium bicarbonate. DW of 0.9% sodium chloride washed samples was more than 0.8 times higher than the other three washed samples. In most of the cases, there was no significant difference between DW of samples washed by ammonium formate and ammonium bicarbonate solutions (p>0.05). The AFDW of the non-washed algal samples was about twice that washed samples, and could be accounted for by volatile component in the sea water medium. Isotonic solution of ammonium bicarbonate is a satisfactory washing agent for algal cells for dry weight determination. This revised version was published online in September 2006 with corrections to the Cover Date.     

Moisture distribution and texture of spaghetti rehydrated under different conditions

Dried spaghetti was rehydrated to its optimal cooking state, known as al dente, at 60, 80, and 100 °C, in distilled water or 0.1, 1.0, and 2.0 mol/L sodium chloride solutions. Then, the moisture distributions and stress–strain curves were examined to identify the major factors governing the texture of rehydrated spaghetti. The difference in moisture content between the inner and peripheral regions of rehydrated spaghetti and its breaking stress were greater at higher rehydration temperatures; however, rehydration temperature did not affect breaking strain. The sodium chloride concentration of the immersion solution did not affect moisture distribution or breaking stress, while breaking strain was decreased by rehydration at higher sodium chloride concentrations. The results obtained in this study suggest that moisture distribution within spaghetti and its material properties govern its breaking stress and strain, respectively.     

Use of a protective solution in the inoculation of bacterial cultures

The influence of the composition of solutions used for growing the bacterial cultures on the survival of microorganisms has been studied. The use of a protective solution, consisting of sodium chloride, orthophosphates, magnium sulfate, and gelatin, for suspensions of bacterial cultures has provided a higher isolation rate of enteric microorganisms, vs. cultivation with 0.9% sodium chloride solution. In evaluating the sensitivity of thioglycol medium test cultures are recommended to be inoculated with the above protective solution.     

Standard electromotive force of the H2-AgCl;Ag cell in 30, 40, and 50 mass% glycerol/water from −20 to 25 °C: pK2 and pH values for a standard “Mops” buffer in 50 mass% glycerol/water

Data are presented regarding the establishment of the pH (designated pH^*) of a standard buffer solution suitable as a pH reference in 50 mass% glycerol/water mixtures at temperatures ranging from −20 to 25 °C. The buffer material selected was the ampholyte Mops [(3-N-morpholino)-propane sulfonic acid], and the reference standard consists of equal molal amounts of Mops and its sodium salt. The assignment of pH^* values is based on measurements of the electromotive force (emf) of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦ Mops, Na Mopsate, NaCl ¦ AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Mops)^±/ah (Mopsate)⁻ + H ⁺. The standard emf of the silver-silver chloride electrode in 30, 40, and 50 mass% glycerol/water mixtures was determined from emf measurements of the cell at subzero temperatures with HCl solutions replacing the buffer-chloride mixtures.     

Effect of salt concentration and temperature on survival of Legionella pneumophila

The effects of various concentrations of sodium chloride solutions (0·1%–3%) and different temperatures (4, 10, 20, 30 and 37 °C) on survival of Legionella pneumophila were investigated. It was found that at temperatures between 4 °C and 20 °C, Legionella organisms survived in salt solutions up to 3% NaCl. Only the combination of high temperatures, i. e. 30 °C and 37 °C, with NaCl concentrations over 1·5%, reduced cell numbers significantly. It was interesting to note that the addition of small amounts of NaCl (0·1%–0·5%) enhanced survival of Leg. pneumophila , suggesting a protective effect of NaCl. In order to obtain information about conditions encountered in the environment, the survival experiments were repeated in sterile sea water from the Baltic Sea and the North Sea. The marked bacterial die-off, especially at higher temperatures, was not observed in natural sea water. All these results indicate that Leg. pneumophila can survive in the marine environment.     

Relationship between preferential interaction of a protein in an aqueous mixed solvent and its solubility

The present paper is devoted to the derivation of a relation between the preferential solvation of a protein in a binary aqueous solution and its solubility. The preferential binding parameter, which is a measure of the preferential solvation (or preferential hydration) is expressed in terms of the derivative of the protein activity coefficient with respect to the water mole fraction, the partial molar volume of protein at infinite dilution and some characteristics of the protein-free mixed solvent. This expression is used as the starting point in the derivation of a relationship between the preferential binding parameter and the solubility of a protein in a binary aqueous solution. The obtained expression is used in two different ways: (1) to produce a simple criterion for the salting-in or salting-out by various cosolvents on the protein solubility in water, (2) to derive equations which predict the solubility of a protein in a binary aqueous solution in terms of the preferential binding parameter. The solubilities of lysozyme in aqueous sodium chloride solutions (pH=4.5 and 7.0), in aqueous sodium acetate (pH=8.3) and in aqueous magnesium chloride (pH=4.1) solutions are predicted in terms of the preferential binding parameter without any adjustable parameter. The results are compared with experiment, and for aqueous sodium chloride mixtures the agreement is excellent, for aqueous sodium acetate and magnesium chloride mixtures the agreement is only satisfactory.     

Effects of physico-chemical factors influencing tyramine production by Carnobacterium divergens

Tyramine production by a strain of Carnobacterium divergens was tested in relation to different conditions of pH, temperature, glucose, oxygen availability, potassium nitrate and sodium chloride content, using a combination of a Doehlert and Plackett-Burman experimental design. A second degree polynomial model was chosen to describe tyramine production which was quantified by high performance liquid chromatography. Maximal tyramine production occurred during the stationary phase in acidic conditions obtained by low initial pH (<5) or addition of glucose (0·6%) to the medium. Production was slower at 5°C than at 23°C and 10% sodium chloride inhibited this production. However, the formation of tyramine was not affected by the presence of potassium nitrate or oxygen availability.     

New methods allowing the detection of protein aggregates

Aggregation compromises the safety and efficacy of therapeutic proteins. According to the manufacturer, the therapeutic immunoglobulin trastuzumab (Herceptin®) should be diluted in 0.9% sodium chloride before administration. Dilution in 5% dextrose solutions is prohibited. The reason for the interdiction is not mentioned in the Food and Drug Administration (FDA) documentation, but the European Medicines Agency (EMEA) Summary of Product Characteristics states that dilution of trastuzumab in dextrose solutions results in protein aggregation. In this paper, asymmetrical flow field-flow fractionation (FFF), fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy (TEM) have been used to characterize trastuzumab samples diluted in 0.9% sodium chloride, a stable infusion solution, as well as in 5% dextrose (a solution prone to aggregation). When trastuzumab samples were injected in the FFF channel using a standard separation method, no difference could be seen between trastuzumab diluted in sodium chloride and trastuzumab diluted in dextrose. However, during FFF measurements made with appropriate protocols, aggregates were detected in 5% dextrose. The parameters enabling the detection of reversible trastuzumab aggregates are described. Aggregates could also be documented by fluorescence microscopy and TEM. Fluorescence spectroscopy data were indicative of conformational changes consistent with increased aggregation and adsorption to surfaces. The analytical methods presented in this study were able to detect and characterize trastuzumab aggregates.     

An approach to the study of phase separation in ternary aqueous systems

1. Simple thermodynamic expressions are used to describe the properties of uncharged binary and ternary polymer solutions, in particular the sedimentation equilibrium of binary systems and the osmotic pressures and `incompatible' phase separations of ternary systems. 2. Sedimentation-equilibrium experiments were performed on four samples of dextran and two of polyethylene glycol. The critical points of the phase diagrams were determined for the mixed solutions of polyethylene glycol–dextran–water and of polyethylene glycol–bovine serum albumin–0·2m-sodium chloride solution. Osmotic pressures were measured on a single-phase mixed solution of a polyethylene glycol and a dextran. By use of the simple thermodynamic expressions consistent values of second virial and interaction coefficients for the materials used were obtained from these experiments. 3. The interpretation of the values of the second virial and interaction coefficients, on the basis of three models of molecular interaction, is discussed.     

A Note on the Survival of some Bacteria in Different Diluents

The best diluent for four bacterial species was 0·1% (w/v) peptone solution. Tap water containing 0·1% (w/v) sodium thiosulphate was less satisfactory but tap water, tap water treated with charcoal, quarter-strength Ringer's solution, 0·85% (w/v) sodium chloride solution and glass distilled water were all bactericidal to one or more of the test species.