Phase equilibria and gelation in gelatin/maltodextrin systems — Part IV: composition-dependence of mixed-gel moduli

The storage moduli (G′) of phase-separated co-gels formed by quench-cooling mixed solutions of gelatin and potato maltodextrin (Paselli SA-2 and SA-6) have been related quantitatively to the experimentally-determined concentration-dependence of G′ for the constituent polymers. Distribution of water between the phases was examined explicitly by using polymer blending laws to derive calculated moduli for gelatin-continuous and maltodextrin-continuous networks over the entire range of solvent partition. Allowance was made for the direct contribution of polymer chains, and for density differences between the phases, in calculating relative phase-volumes. The effect of gel formation prior to phase-separation was calculated using classical theory for network de-swelling. Good agreement with observed moduli for more than 30 gelatin/maltodextrin combinations was achieved using a single adjustable parameter, p (the ratio of solvent to polymer in the gelatin phase divided by the same ratio for the maltodextrin phase), with an optimum value of p ≈ 1·8 for both SA-6 and SA-2.     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part II: polymer incompatibility in solution

The effect of thermodynamic incompatibility in mixed solutions of gelatin and Paselli maltodextrins SA-6 and SA-2 has been studied at a temperature (45°C) where the individual polymers are stable as disordered coils. Concentrated mixtures of SA-6 and gelatin showed classic phase separation into two co-existing liquid layers, with compositions lying along a well-defined binodal. On decreasing SA-6 concentration below the binodal, however, a substantial proportion (up to 60%) of the maltodextrin was precipitated, with normal single-phase solutions occurring only at much lower concentrations of both polymers. SA-2 showed a more extreme version of the same behaviour, with precipitation of up to 100% of the maltodextrin and no evidence of co-existing liquid phases at any accessible concentrations. In both cases, the amount of maltodextrin precipitated was proportional to the square of its initial concentration and to the first power of gelatin concentration, indicating that gelatin drives self-association and aggregation of maltodextrin when both polymers are present in a single liquid phase. ¹H NMR showed the precipitated maltodextrin to be higher in molecular weight and in degree of branching than the material remaining in solution, and particlesize analysis indicated that the volume of the individual maltodextrin particles increased linearly with the total mass precipitated.     

Phase separation and gel formation in kinetically trapped gelatin/maltodextrin gels

The kinetics of phase separation and gel formation of gelatin/maltodextrin mixtures have been studied using confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), stereological image analysis and rheology. The quantified microstructural parameters were the volume-weighted mean volume and the interfacial area. The temperature of phase separation was defined as the temperature where the first signs of phase separation were observed in CLSM. The gelatin concentration varied between 4 (wt.) and 5% and the maltodextrin concentration varied between 2 and 6%. Maltodextrin was labelled covalently with RITC to improve the contrast between the gelatin phase and the maltodextrin phase. The solutions were cooled from 60 to 10 degrees C, and the cooling rates used were 0.4, 1 and 3 degrees C/min. All systems were found to be gelatin continuous under the experimental conditions used. The results showed that the temperature of phase separation (TPS) increased both with the gelatin concentration and the maltodextrin concentration, but particularly with the former. The size of the maltodextrin inclusions increased with TPS, and the interfacial area decreased with increasing TPS. The diameter of the maltodextrin inclusions varied between 1.6 and 8.5 microm at 1 degrees C/min. The size of the maltodextrin inclusions was found to increase with decreasing cooling rate and was largest at 0.4 degrees C/min. The TPS was compared with the gelation temperature (Tgel) at three different concentrations of gelatin and maltodextrin (4/3, 4/5, 5/5%). CLSM micrographs and TEM micrographs showed that these three concentrations of gelatin and maltodextrin had different microstructures. At a TPS above Tgel (5/5%), the phase separation proceeded faster than the gel formation and the microstructure had few, large maltodextrin inclusions and a clean continuous gelatin phase. At a TPS comparable with Tgel (4/5%), phase separation occurred during gel formation, which led to a varying microstructure and competition between the phase separation and the gel formation. At a TPS below Tgel (4/3%), gel formation proceeded faster than the phase separation and the microstructure had many, small inclusions and a diffuse microstructure, and the phase separation was incomplete. It was established that the microstructure was determined by the relative rates of the phase separation and the gel formation. Three different zones of phase separation could be distinguished based on comparisons of TPS and Tgel, and results from CLSM, TEM and image analysis.     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part I: gelation of individual components

As a prelude to studies of co-gelation with galatin, the gelation behaviour of Paselli maltodextrins SA-6 and SA-2 (DE ≈ 6 and 2, respectively) was mapped out over the experimentally-accessible range of temperature (T) and concentration (c), using a simple visual method to determine the time required for formation of a self-supporting network (t_g). For both samples, log t_g decreased linearly with log c and increased linearly with T. At equivalent temperatures and concentrations, SA-2 gelled between 20 and 60 times faster than SA-6.

Selected samples were monitored more rigorously by mechanical spectroscopy, taking t_g as the time at which elastic response (G′) became greater than viscous response (G″). In all cases the values of t_g obtained by this procedure were lower than those from visual inspection, by a constant factor of about 3·4.

The concentration-dependence of gel moduli (G′) for SA-2 and for gelatin (second-extract limed ossein; LO-2) fitted accurately to the form anticipated from cascade theory for normal polymer networks. For SA-6, by contrast, log G′ varied linearly with log c over the entire range at which measurements could be made, indicating a different mechanism of structure-formation (such as the agglomeration of short, aggregated helices).     

Some physical and microstructural properties of genipin-crosslinked gelatin-maltodextrin hydrogels

The physical properties and microstructure of gelatin-maltodextrin hydrogels fixed with genipin (GP) were investigated as a function of pH (3-7), maltodextrin (MD) (0-9%, w/w) and GP (0-10 mM levels), at a constant gelatin (G) concentration (10%, w/w). Network strength (elastic modulus, E) and swelling behavior were characterized by large deformation testing and by swelling index (SI). In general, network strength increased and swelling decreased at higher pH, MD and GP levels, except at pH 3, where E was independent of the GP concentration until approximately 7.5 mM, above which it declined. Confocal scanning laser microscopy (CLSM) images showed phase separation to be suppressed at pH 3, whereas at pH 7, separation into a self-similar dispersed phase was apparent. Overall, the judicious use of GP to crosslink G was an appropriate means of kinetically trapping MD within the gelatin network.     

Viscoelastic and Thermal Properties of Collagen–Xanthan Gum and Collagen–Maltodextrin Suspensions During Heating and Cooling

The purpose of this work was to investigate the viscoelastic properties of aqueous suspensions of crude collagen powder extracted from bovine hides and nonsubmitted to the hydrolysis reaction that leads to gelatin. The studied variables included the collagen concentration and the addition of xanthan gum or maltodextrin at varied concentrations during heating/cooling of the mixtures. Differential scanning calorimetry thermograms showed that the addition of polysaccharides decreased the endothermic peak areas observed at the denaturation temperature of collagen. The rheological properties of the pure collagen suspensions were highly dependent on concentration: 4% and 6% collagen suspensions presented a great increase in the storage modulus after heating/cooling, whereas for concentrations of 8% and 10% G′ decreased during heating and did not recover its original value after heating/cooling. The frequency sweeps showed that the thermal treatment was responsible by the strengthening of the interactions that formed the polymer network. Addition of 0.1% xanthan gum to collagen suspensions increased the gel strength, especially after heating/cooling of the system, whereas increasing gum concentration to 0.3% resulted in a weaker gel, which could indicate thermodynamic incompatibility between the biopolymers. Mixtures of collagen and maltodextrin resulted in more fluid structures than those obtained with pure collagen at the same collagen concentration and the range of temperatures in which these mixtures behaved as a gel decreased with increasing concentrations of both collagen and maltodextrin, suggesting incompatibilities between the biopolymers.     

Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections

We used a model system to study whether measurements of absolute local antigen concentrations at the electron microscopic level are feasible by counting immunogold labeling density in ultra-thin sections. The model system consisted of a matrix of a variable concentration of gelatin, which was mixed with given concentrations of rat pancreas amylase and fixed according to various fixation protocols. With a relatively mild fixation, there was no clear proportionality between anti-amylase gold labeling and amylase concentration in ultra-thin cryosections. This was presumably due to uncontrolled loss of amylase from the sections. After stronger fixation with 2% glutaraldehyde for 4 hr, labeling density reflected the amylase concentration very well. We observed that matrix (gelatin) density influenced labeling density. A low gelatin concentration of 5% allowed penetration of immunoreagents into the cryosection, resulting in a high and variable labeling density. In gelatin concentrations of 10% and 20%, labeling density was lower but proportional to amylase concentration. To establish an equal (minimal) penetration of immunoreagents, we embedded model blocks with different matrix densities in polyacrylamide (PAA). In ultra-thin cryosections of these PAA-embedded blocks, anti-amylase labeling was proportional to amylase concentration even at a low (5%) gelatin concentration. Anti-amylase labeling in ultra-thin sections from Lowicryl K4M low temperature-embedded blocks was higher than in PAA sections, but the results were less consistent and depended to some extent on matrix density. These results, together with the earlier observation that acrylamide completely penetrates intracellular compartments (Slot JW, Geuze HJ: Biol Cell 44:325, 1982), demonstrate that it is possible to measure true intracellular concentrations of soluble proteins in situ using ultra-thin cryosections of PAA-embedded tissue.     

Opposing effects of NaCl on reversibility and thermal stability of halophilic beta-lactamase from a moderate halophile, Chromohalobacter sp. 560

Beta-lactamase from a moderately halophilic organism is expected to show salt-dependent stability. Here we examined the temperature-dependence of stability at different salt concentrations using circular dichroism (CD) and enzyme activity. NaCl showed opposing effects on melting temperature and reversibility of the thermal melting. Increasing NaCl concentration greatly increased the melting temperature from, e.g., 41 degrees C in the absence of NaCl to 61 degrees C in 3 M NaCl. Conversely, reversibility decreased from 92% to 0% in the corresponding NaCl solutions. When beta-lactamase was heated at different temperatures and NaCl concentrations, the activity recovery followed the reversibility, not the melting temperature. Heating beta-lactamase at 63 degrees C, slightly above the onset temperature of melting in 2 M NaCl and far above the melting in 0.2 M NaCl, showed a much greater recovery of activity in 0.2 M NaCl than in 2 M NaCl, again consistent with the reversibility of melting.     

Butanol Production by a Butanol-Tolerant Strain of Clostridium acetobutylicum in Extruded Corn Broth

By employing serial enrichment, a derivative of Clostridium acetobutylicum ATCC 824 was obtained which grew at concentrations of butanol that prevented growth of the wild-type strain. The parent strain demonstrated a negative growth rate at 15 g of butanol/liter, whereas the SA-1 mutant was still able to grow at a rate which was 66% of the uninhibited control. SA-1 produced consistently higher concentrations of butanol (from 5 to 14%) and lower concentrations of acetone (12.5 to 40%) than the wild-type strain in 4 to 20% extruded corn broth (ECB). Although the highest concentration of butanol was produced by SA-1 and the wild-type strain in 14% ECB, the best solvent ratio with respect to optimizing butanol and decreasing acetone occurred between 4 and 8% ECB for SA-1. SA-1 demonstrated higher conversion efficiency to butanol than the wild-type strain at every concentration of ECB tested. Characterization of the wild-type and SA-1 strain in 6% ECB demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, carbohydrate utilization, pH resistance, and final butanol concentration in the fermentation broth.     

Effects of Polyols and Sugars on the Sol-Gel Transition of Gelatin

The melting temperature of gelatin gel increased with increasing concentrations of polyols and sugars added at any gelatin concentation. The enthalpy of gel melting measured by Eldridge-Ferry plots and calorimetric measurements was decreased by addition of these polyhydric compounds, indicating that the gel stabilization by them is predominantly due to the large decrease in entropy of gel melting. Circular dichroism analysis showed that the helix formation of gelatin molecules was enhanced with increasing concentrations of these additives, but there was no positive correlation between their helix-forming ability and gel-stabilizing ability. The viscosity of gelatin solution was less in these mixed solvents than in water. Based on these results, a possible stabilization mechanism of gelatin gel by polyhydric compounds was discussed, in comparison with that of native collagen, in terms of the thermodynamics of protein-solvent interactions.     

Texture and Microstructure of Gelatin/Corn Starch-Based Gummy Confections

Texture of gummy gels prepared with gelatin and acid modified corn starch (AMCS) was quantified by instrumental techniques and the gel microstructure was examined by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Gelatin:AMCS gummy gels were divided in two groups: Group 1, containing different gelatin (0?C10?wt%) and AMCS (0?C10?wt%) concentrations, totalizing 10?wt% solids; and Group 2, which was prepared with a fixed gelatin concentration (8?wt%) and varied AMCS concentrations (0?C5?wt%). All gummy gels were formulated with maltitol syrup and xylitol, shaped in cylindrical molds and submitted to instrumental texture profile analysis (TPA) tests and colorimetric analysis. Group 1 pure starch gels (10?% AMCS) presented the highest stringiness and adhesiveness. In samples of Group 2 the introduction of AMCS dramatically changed the structure of the gelatin gels. Thermodynamic incompatibility was evident even at the lowest AMCS concentration. Moreover, increasing AMCS concentrations lead to an increase in the number of hollow zones including starch granules inside them. In addition, introduction of AMCS in the samples of Group 2 caused an increase in hardness and opacity and a decrease in stringiness and adhesiveness. On the other hand, results from TPA tests showed that the addition of AMCS to gelatin gels in suitable proportions can be a feasible alternative in the formulation of gummy confections.     

Immunogold determination of amylase concentrations in pancreatic subcellular compartments

We used the immunogold method on ultrathin cryosections to measure intracellular amylase (Am) concentrations in subcellular compartments of rat exocrine pancreatic cells. Previously, the quantitation procedure was characterized in a model system consisting of Am dispersed at known concentration in a matrix of gelatin. Variations in labeling efficiency, due to differences in matrix density, were equalized by embedding in 30% polyacrylamide (PAA). Here we applied these model conditions to rat pancreas and established intracellular Am concentrations [Am]. Specimen blocks were composed of tissue and a reference layer of gelatin mixed with a known Am concentration ([Am]r), both fixed in glutaraldehyde. Cryosections of the PAA embedded blocks were immunogold labeled for Am. The labeling density was measured in the reference layer (LDr) and in structures in exocrine cells that were involved in Am synthesis and transport (LDs). In each of these structures the Am concentration ([Am]s) was calculated from: [Am]s = [Am]r. LDs/LDr In this way we measured average concentrations ranging from 63 mg/ml in rough endoplasmic reticulum to 261 mg/ml in secretory granules. Concentration of Am appeared to occur mainly in the most cis- and the most trans-Golgi cisternae. To check whether sterical hindrance was an inherent bias to the [Am] measurements in compartments that contained high concentrations of the enzyme, the labeling efficiency for Am in intact isolated secretory granules in gelatin and embedded in PAA, was compared with the efficiency when the granules were lysed and approximately 50 times diluted in gelatin before PAA embedment. It appeared that Am was detected with similar efficiency under both conditions. This demonstrated that sterical hindrance did not cause errors in the measurements of cellular Am concentrations.     

FTIR microspectroscopy study of composition fluctuations in extruded amylopectin-gelatin blends

The spatial variation in the composition of nonexpanded biopolymer blends prepared by extrusion of mixtures of gelatin with either native or pregelatinized waxy maize starch was studied using a 30-microm aperture FTIR microspectroscopy technique. The ratio of the areas of the "saccharide" bands (953-1180 cm(-1)) and the amide I and II bands (1483-1750 cm(-1)) was used to monitor the relative distributions of the two components of the blend. Two calibration methods were used to obtain amylopectin concentration values from the ratios of the IR bands. The results suggested a high degree of heterogeneity in these blends, despite the thorough mixing expected by twin-screw extrusion processing. The concentration fluctuations were greater for the blends produced by extruding gelatin and native waxy maize starch mixtures. This was in agreement with the reduced degree of conversion of the starch granules when extruded in the presence of gelatin. The FTIR 2-dimensional maps obtained suggested that in the blends produced from either native or pregelatinized starch at all concentrations studied (25/75, 50/50, and 75/25 amylopectin/gelatin) the gelatin constituted the continuous phase. The effect of the spatial resolution on the FTIR microspectroscopy results was considered and the proposed interpretation was verified by the use of polarized light microscopy and FTIR microspectroscopy acquired at higher spatial resolution (10 microm).     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.     

Protein adsorption at solid-liquid interfaces: Part II--Adsorption from binary protein mixture

Extent of adsorption of proteins at alumina-water interface from solutions containing binary mixture of beta-lactoglobulin and bovine serum albumin (BSA), beta-lactoglobulin and gelatin, and gelatin and bovine serum albumin has been estimated as functions of protein concentrations at varying pH, ionic strength, temperature and weight fraction ratios of protein mixture. The extent of adsorption (gamma lacw) of lactoglobulin in the presence of BSA increases with increase of protein concentration (Clac) until it reaches a maximum but a fixed value gamma lacw(m). Extent of adsorption gamma serw also initially increases with increase of protein concentrations until it reaches maximum value gamma serw(m). Beyond these protein concentrations, adsorbed BSA is gradually desorbed due to the preferential adsorption of lactoglobulin from the protein mixture. In many systems, gamma serw at high protein concentrations even becomes negative due to the strong competition of BSA and water for binding to the surface sites in the presence of lactoglobulin. For lactoglobulin-gelatin mixtures, adsorption of both proteins is enhanced as protein concentration is increased until limiting values for adsorption are reached. Beyond the limiting value, lactoglobulin is further accumulated at the interface without limit when protein concentration is high. For gelatin-albumin mixtures, extent of gelatin adsorption increases with increase in the adsorption of BSA. The limit for saturation of adsorption for gelatin is not reached for many systems. At acid pH, adsorbed BSA appears to be desorbed from the surface in the presence of gelatin. From the results thus obtained the role of electrostatic and hydrophobic effects in controlling the adsorption process has been analysed.     

Effect of galactomannan addition on the thermal behaviour of κ-carrageenan gels

Gelation/melting cycles of κ-carrageenan/galactomannan (guar, tara and locust bean gums) binary systems have been studied by measuring dynamic rheological parameters. Two experimental conditions were used, (i) the total polysaccharide concentration was kept at 1% and the κ-carrageenan/galactomannan ratio fixed at 4:1 and (ii) the κ-carrageenan concentration was fixed at 0·75% and the galactomannan content varied from 0% to 1·2%. A thermal hysteresis was observed for all mixed systems and was found to depend on the galactomannan used. From a comparison of the gelation temperature (T_g) and melting temperature (T_m) to values obtained with κ-carrageenan alone, it was suggested that galactomannan interferes with gel structure by the formation of a secondary network provided that the M/G ratio is high enough.     

Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation

A new cell cryopreservation strategy for cell-assembling constructs was proposed. With this strategy, different concentrations of dimethysulfoxide (DMSO) and dextran-40 were directly incorporated into the cell/gelatin/alginate systems, prototyped according to a predesigned structure, cryopreserved at −80 °C for 10 days and followed a thawing process at 17 °C. The rheological properties, bonding water contents and melting points of the gelatin/alginate hydrogel systems were changed with the addition of different amounts of DMSO. The microscopy analysis, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) and hematoxylin and eosin (HE) staining indicated that the cell numbers were progressively in a selected DMSO concentration range. With DMSO 5% (v/v) alone, the metabolic rate in the construct attained (81.3 ± 5.7)%. A synergistic effect was achieved with the combination of the DMSO/gelatin/alginate and dextran-40/gelatin/alginate hydrogel systems. These results indicated that the inclusion of DMSO and dextran-40 in the hydrogel could effectively enhance the cell preservation effects. This cryopreservation strategy holds the ability to be widely used in organ manufacturing techniques.     

The properties of gelatin-poly (gamma-glutamic acid) hydrogels as biological glues

The influence of the molecular weight and the type of gelatin (A or B), as well as the molecular weight of poly (gamma-glutamic acid) (gamma-PGA), on the properties of gelatin/gamma-PGA mixed bioadhesives were studied. The gelation of the system was enhanced by a crosslinker, 1-(3-dimethylaminopropyl)-3-(ethylcarbodiimide) hydrochloride (EDC). The gelation time of the bioadhesives was analyzed using rheological measurements. The results indicated that the type of gelatin was a critical factor in determining the gelation time of the biological glues. The mixed glues had greater bonding strength and smaller gelation times as the molecular weight of gamma-PGA or gelatin increased. The swelling ratio decreased and the denaturation temperature increased upon raising the EDC concentration, indicating a greater degree of crosslinking at higher EDC concentrations. The mixed glues crosslinked with various concentrations of EDC (1.7-2.5%) showed no cytotoxicity to fibroblasts. In addition, no significant inflammatory response was observed in the rat subcutaneous implantation. The bioadhesives based on gelatin/gamma-PGA remained at the site for 7 days while the fibrin glue had almost completely degraded. By choosing the appropriate gelatin type and higher molecular weight gamma-PGA in the mixtures, the gelatin/gamma-PGA biological glues could serve as soft tissue adhesives. Rheological characterization was essential in the evaluation of biological glues.     

Structural properties of pectin-gelatin gels. Part II: effect of sucrose/glucose syrup

Small deformation dynamic oscillation and bright field microscopy were used to examine the structural properties of single and mixed high methoxy pectin and gelatin systems in the presence of sucrose/glucose syrup blends. Co-solute concentrated (≥78%) systems of the polysaccharide form rubbery structures which are readily transformed into glassy consistencies according to the time-temperature superposition principle. Increasing amounts of co-solute in the gelatin samples induce changes in viscoelasticity from that of conventional hydrogels to mechanical traces that cover much of the plateau region and the beginning of the glass transition area. Furthermore, manipulation of the protein/ sugar ratio can result in strong crystalline matrices, or viscoelastic solutions where the co-solute forms the continuous phase and the gelatin inclusions can undertake a conformational transition. The properties of the single components were used to rationalise the phase behaviour of their mixtures. Upon triggering the gelation of pectin, mixtures can be made where either gelatin or both components form a continuous phase. Results are discussed in the light of evidence obtained from the ethylene glycol work in Part I.     

Stabilization of configurational states and enzyme activities in subcellular fractions after fixation with extremely low concentrations of glutaraldehyde

Synopsis The activities of various enzymes in some subcellular organelle fractions were examined after fixation in glutaraldehyde of various concentrations. A high speed centrifuge was used to shorten the fixation time.At the lowest concentration (0.01%) glutaraldehyde stabilized instable configurational states of mitochondria as revealed by electron microscopy. In addition, at this concentration, at least 70% of the original monamine oxidase, ATPase and cytochrome oxidase activities were preserved. The activity of acid phosphatase, on the other hand, was enhanced in a lysosomal fraction when fixed with the aldehyde at higher concentrations, e.g. 0.1% and 1.0%. It is possible that the aldehyde at higher concentrations has the same effects on the lysosomal membrane as freeze-thawing. Glucose-6-phosphatase activity was well-preserved in a microsomal fraction fixed with 0.01% glutaraldehyde but was decreased drastically when the concentration of the aldehyde was greater than 0.05%.