Kinetics Of Enzyme Release Research Articles

Comparative Investigation of pH-Dependent Availability of Pancreatic Enzyme Preparations In Vitro.

This study aimed to compare different pancreatic enzyme preparations (PEPs) available in Germany regarding particle geometry and size, and to evaluate enzyme activity under physiologically relevant conditions in vitro. Pancreatic endocrine insufficiency is characterized by deficiency of pancreatic enzymes resulting in maldigestion. It is orally treated by pancreatic enzyme replacement therapy. The formulations differ in their physical properties and enzyme release behavior, potentially resulting in inconsistent dosages and poor interchangeability of products. A total of 25 products were analyzed for particle size and number of particles per capsule. Enzyme activities of lipase, amylase, and protease were measured by digestion of olive oil emulsion, starch, and casein, respectively. To analyze enzyme release, gastric environments were simulated by incubating PEPs at pH 1, 4, or 5. Duodenal conditions were simulated by subsequent incubation at pH 6. Regarding physical properties and enzyme release kinetics, considerable differences between different PEPs were found. Furthermore, compared to the label claim, excess lipase activity was observed for most products, reaching up to 148%. These in vitro results suggest poor interchangeability of PEPs, potentially explained by physical and release characteristics. Physicians and patients should be aware of the potential gap between label claims and the real-life performance of different PEPs.

CREATION AND PROPERTIES OF BIOCOMPOSITE NANOPARTICLES BASED ON FUCOIDAN AS CARRIERS OF FIBRINOLYTIC ENZYME

Magnetically guided biodegradable nanocomposites are a new class of functional materials that expand the possibilities of clinical therapy. The paper describes the chemistry of the formation of a nanobiocomposite material based on a natural polymer synthesized by electrostatic interaction of a polysaccharide and magnetite in an aqueous medium. The possibility of electrostatic immobilization of a model fibrinolytic enzyme was studied. An increase in the electrochemical potential of particles during immobilization of a fibrinolytic enzyme was established by the method of dynamic light scattering. The degree of enzyme inclusion was 9.95±0.05 mass%. The average particle size of the nanobiocomposite material after immobilization of the enzyme was 264.3 nm. The study of the magnetic properties showed the absence of residual magnetization, which makes it possible to exclude the possibility of magnetic aggregation when using the biocomposite in a biological application. The saturation magnetization of the formed material was 57.1 ± 2.3 kA/m, which is lower than that of pure magnetite sol (MS ∼ 73.8 ± 3.4 kA/m), but sufficient for manipulation under conditions of resistance to blood flow. The kinetics of the release of an enzyme preparation in a phosphate buffer from a nanocomposite carrier based on fucoidan was studied. To study the kinetics of enzyme release by mathematical modeling, zero and first order models, the Korsmeier-Peppas model, the Higuchi model, and the Baker-Lonsdale model were used. It has been established that the kinetics of fibrinolytic enzyme release is described with high accuracy (r2 = 0.98) by the Korsmeier–Peppas equation, where the process of release of the enzyme preparation is influenced by diffusion obeying Fick's law. It has been established that zero and first order models are applicable only to the initial (0-40 min, where r2 = 0.96) and final (80-320 min, r2 = 0.93) stage of release.

RELEASE OF INTRACELLULAR β-GALACTOSIDASE FROM LACTOBACILLUS ACIDOPHILUS AND L-ASPARAGINASE FROM PECTOBACTERIUM CAROTOVORUM BY HIGH-PRESSURE HOMOGENIZATION

This article reports a comparison of the ease of disruption of gram-positive (Lactobacillus acidophilus) and gram-negative (Pectobacterium carotovorum) bacteria using high-pressure homogenization (HPH). The location factor for both enzymes calculated from enzyme release kinetics together with localization studies identified them as cytoplasmic enzymes. The results showed that release of β-galactosidase by HPH of L. acidophilus was more difficult than the release of L-asparaginase from P. carotovorum. It took nine passes at 55.14 MPa for maximum release of β-galactosidase (0.949 IU/mL) as compared to six passes at 41.35 MPa for L-asparaginase (4.653 IU/mL); 1.7 IU of β-galactosidase was released as against 11.5 IU of L-asparaginase per MJ of energy during high-pressure homogenization.

Effect of Calcium Phosphate Addition to Alginate Microspheres: Modulation of Enzyme Release Kinetics and Improvement of Cell Adhesion

In this study, the addition of calcium phosphate powders to an alginate matrix was evaluated as a strategy to modulate enzyme release-kinetics from alginate microspheres and, simultaneously, to improve cell adhesion to the polymer. Pre-adsorption of the enzyme to the ceramic powders resulted in a more adequate release pattern. The ratio of ceramic-to-polymer had a pronounced effect on osteoblast adhesion to microspheres. Cells were only able to spread on microspheres with the highest percentage of ceramic (0.4 w/w using a 1.5% w/v alginate solution).

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O2- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O2- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O2- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O2- production and MPO release from stimulated neutrophils.

CREATINE KINASE SUBFORMS IN MARATHON RUNNERS 843

The lack of a rapid diagnostic marker specific to acute myocardial infarction (AMI) led to the development of a new high-voltage electrophoresis assay that separates creatine kinase (CK)-MB isoenzyme into its subforms. MB1 and MB2. The ability of the assay to detect early increases in the MB2/MB1 ratio in the blood and to reliably detect AMI within 6 hrs after the onset of chest pain has been shown to provide improved diagnosis of AMI. Positive clinical diagnosis is met when both the MB2 levels and subform ratios are positive (MB2≥2.6 U/L and ratio ≥ 1.7). Limited research has been conducted on the effectiveness of this assay on physically active individuals. Thus, the purpose of this study was to investigate the temporal patterns of MB subforms in plasma of marathon runners. Blood samples were obtained from 33 subjects (22 men, 11 women) prior to, immediately post, and 2.4, and 6 hrs post marathon race. Plasma MB2 activity, MB1 activity, MB2/MB1 ratio. CK-MB, and relative index (CK-MB/total CK) prior to the race were all within normal. Plasma MB2 activity, MB1 activity, and MB2/MB1 ratio immediately post race (3 to 5 hrs after race start) were elevated to clinically abnormal levels. Ninety% (30/33), 94% (31/33) and 100% of the subjects demonstrated a combined positive MB2 level and subform ratio immediately post, and 2 and 4 hrs post race. The kinetics of enzyme release in marathon runners is similar to that previously reported for AMI patients;acute acute skeletal muscle injury results in the release of tissue subforms of MB approx. 3 hrs after the onset of injury. These data suggest that the MB2/MB1 ratio cannot be used to differentiate between damage to the skeletal muscle vs the myocardium. Relative index values (<5%) across time suggest that skeletal muscle is the site of injury because values are less than that previously reported in AMI.

An experimental investigation of enzyme release from poly(vinyl alcohol) crosslinked microspheres.

Crosslinked poly(vinyl alcohol) particles were prepared by the addition of glutaraldehyde into a PVA methanol/water solution in the presence of 0.2 N sulphuric acid. The polymer solution was dispersed in mineral oil in a jacketed vessel, with the aid of a six-blade impeller. Spherical crosslinked particles in the size range 30-80 microns were obtained by varying the degree of agitation or/and the amount of suspending agent. The crosslinked particles, after washing and drying, were placed into a protease enzyme solution for loading. The enzyme-containing water-swollen particles were subsequently removed from the solution and the enzyme release kinetics determined by a UV spectrophotometer. The influence of the degree of crosslinking, ionic strength, pH, particle size, and degree of hydrophilicity of the polymer on the enzyme activity was retained during the adsorption-desorption studies. The release behaviour of enzymes from crosslinked PVA particles exhibited a biphasic kinetic model, with an initial fast release followed by a much slower release rate.

Enzyme release kinetics in a cell disruption chamber of a bead mill.

The kinetics of enzyme release from yeast using a bead mill was investigated. The enzymes investigated were invertase, acid phosphatase, alcohol dehydrogenase and alkaline phosphatase. A two-step consecutive process model was proposed to describe the behavior of enzyme release. The model described well the release of both membrane-bound and non membrane-bound enzymes, such as alkaline phosphatase and other enzymes, respectively.

Kinetics of protein release from yeast using enzymatic lysis for selective product recovery

The kinetics of release of four intracellular enzymes from different yeast cell locations using the Differential Product Release (DPR) method has been investigated. The method uses a combination of physical, chemical and biological agents such as lytic enzymes, an osmotic support and a spheroplast stabilizer. Using the DPR technique a wall enzyme, invertase, was released with a very high specific activity in the first step from a breadmaking strain ofS. cerevisiae. Maximum release could be obtained in this step when the incubation time was extended from 60 min to 100 min. Two cytosol enzymes, α-D-glucosidase and alcohol dehydrogenase were released in the second step. Fumarase was released in the third step almost instantaneously after disruption of the mitochondria which reduces considerably, by ca. 1 hour, the total incubation time of DPR. This paper investigates the kinetics of enzyme release during the 3 steps of DPR.

Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization

Porphyromonas gingivalis W50, W83, A7A1-28, and ATCC 33277 were investigated for their abilities to lyse sheep, human, and rabbit erythrocytes. All of the P. gingivalis strains studied produced an active hemolytic activity during growth, with maximum activity occurring in late-exponential-early-stationary growth phase. The enzyme was cell bound and associated with the outer membrane. Fractionation of P. gingivalis W50 localized the putative hemolysin almost exclusively in the outer membrane fraction, with significant hemolytic activity concentrated in the outer membrane vesicles. Ca2+ and Mg2+ ions significantly increased the expression of hemolytic activity. Hemolytic activity was inhibited by proteinase K, trypsin, the proteinase inhibitors Na-P-tosyl-L-lysine chloromethyl ketone and benzamidine, the metabolic inhibitor M-chlorophenyl-hydrazone, and iodoacetate. KCN and sodium azide (NaN3) only partially inhibited P. gingivalis hemolytic activity, while antiserum to whole cells of each of the P. gingivalis strains had a significant inhibitory effect on hemolytic activity. The P. gingivalis W50 hemolysin was inhibited by cysteine, dithiothreitol, and glutathione at concentrations of at least 10 mM; at low concentrations (i.e., 2 mM), dithiothreitol did not completely inhibit hemolytic activity. Heating to temperatures above 55 degrees C resulted in an almost complete inhibition of hemolytic activity. The effect of heme limitation (i.e., iron) on hemolysin production indicated that either limitation or starvation for heme resulted in significantly increased hemolysin production compared with that of P. gingivalis grown in the presence of excess heme.

Kinetics of enzyme release from breadmaking yeast cells in a bead mill

Four intracellular enzymes from two species of breadmaking yeasts- S. cerevisiae and C. boidinii- have been measured as a function of time during its disruption using a bead mill in batch operation. The amount and rate of enzyme released was dependent on its location inside the cell as well as on the kind of yeast. The maximum amount of invertase, a-D-glucosidase, alcohol dehydrogenase and fumarase was obtained at 2,5,10,15 min. respectively for S. cerevisiae. C. boidinii did not show either invertase nor a-D glucosidase activity and the maximum amount of alcohol dehydrogenase and fumarase were reached at 5 and 20 min. respectively.

Lysosomal enzyme release from human monocytes in response to particulate stimuli.

Lysosomal enzyme release from human monocytes was evaluated in response to opsonized zymosan, opsonized sheep erythrocytes, and latex beads. Monocytes were found to release lysosomal enzymes immediately upon challenge with all three phagocytosable particles. Cytochalasin B enhanced beta-glucosaminidase release from mononuclear cells challenged with opsonized zymosan or opsonized red blood cells, but inhibited the response to latex particles. Lysosomal enzyme release was found to be independent of protein synthesis, and in the absence of cytochalasin B required the stimulus to be presented either as a phagocytosable particle or immobilized on a surface. The kinetics of enzyme release and phagocytosis were also examined and found to be different, lending support to the hypothesis that lysosomal enzyme release may be a physiologic response to a biologic stimulus in vivo and not simply an "accidental" consequence of an ongoing phagocytic event.

Lysosomes in human cell cultures: Kinetics of enzyme release from injured particles

A subcellular fraction prepared from a heteroploid human cell line had the characteristics of lysosomes in that it contained sedimentable acid hydrolases which could be solubilized by adding Triton X-100. Appearance of soluble acid phosphatase activity during acid incubation of these lysosomes was characterized by an initial rapid rise with no further increase until the third hour of incubation. In addition, sedimentable activity increased, suggesting an alteration of permeability of lysosomes to substrate. Ultraviolet irradiation accelerated the release of acid phosphatase during a 90 min period but was not characterized by the initial rapid rise of soluble activity observed with acid incubation. The effects of acid incubation and ultraviolet irradiation were temperature dependent, with no significant release of enzyme activity at 4 °C. Lysosomes from cells grown with 1.5 × 10 −4 M prednisolone released less acid phosphatase during acid incubation. Those from cells grown in concentrations of steroids as low as 2 × 10 −6 M were similarly affected, but only during the first two hours of acid incubation. Cells grown with 4 × 10 −4 M prednisolone showed inhibition of net protein synthesis and no enhancement of lysosome stability.