Effect of Copper on the Function of Isolated Porcine Kidneys Stored Using Simple Hypothermia.

Renal ischemia in the peri-transplant period causes a number of changes that adversely affect the initiation of normal vital functions in grafts after transplantation. To minimise the extent of ischemic damage, organs are stored in preservation fluid. The components of the fluid are supposed to ensure stabilisation of the cell cytoskeleton, protect against oxygen free radicals, reduce cell swelling, and ensure endothelial cell integrity. The aim of this study was to analyse the protective effect of Cu2+, as a component of Biolasol preservative fluid, in the prevention of nephron damage occurring during the graft storage period. Analyses of the effectiveness of copper in the presence of prolactin added to Biolasol fluid were also carried out. Forty isolated pig slaughter kidneys were used in the study, avoiding the use of laboratory animals. The kidneys were stored using simple hypothermia. After 2 h and 48 h of graft storage, selected biochemical indicators of renal function were determined in the collected perfusates. The addition of Cu2+ at a dose of 1 µg/L to the composition of Biolasol fluid was found to affect the generation of ischemic damage in the isolated pig kidney. The intensity of the occurrence of these processes is exacerbated by the presence of prolactin at a dose of 0.1 µg/L.

Effect of Manganese on the Efficacy of Preservative Solution in Protecting Isolated Porcine Kidneys.

BACKGROUND: Manganese is a bioelement essential for the proper functioning of the body. Mn2+ is a cofactor for many enzymes, including manganese superoxide dismutase. Together with the enzymes, it participates in cell defense against reactive oxygen species, immune response, and energy production in mitochondria. The aim of this study was to evaluate the effect of manganese added to Biolasol solution on the storage efficiency of isolated pig kidneys. METHODS: Twenty kidneys from Polish Great White pigs were used in the study. Biolasol solution was modified with the addition of Mn2+ at a concentration of 1 µg/L. Grafts were assigned to 2 groups: A/Biolasol (n = 10) and B/Biolasol + Mn2+ (n = 10). The kidneys were washed with the developed solutions, then stored using the static method for 48h under hypothermia (4°C) and washed again. Markers of normal renal function were analyzed in the collected perfusates-aspartate aminotransferase (AST), alanine aminotransferase (ALT), protein, creatinine, urea, and electrolytes. RESULTS: After 48 hours and 30 minutes of renal preservation, activities/concentrations of biochemical parameters in the Biolasol + Mn2+ group increased (ALT: by 33%; AST: by 86%; urea: by 18%; creatinine: 3.25-fold [P < .05]) vs Biolasol. K+ ion concentration was 33% lower in the Biolasol group after 48 hours and 30 minutes (P < .05) vs group B. The presence of manganese in the composition of Biolasol solution influenced the maintenance of optimal Na+ ion concentration. CONCLUSIONS: Manganese added to the Biolasol solution formulation at a dose of 1 µg/L showed low efficacy in protecting nephrons. We do not recommend adding manganese to the preservative solution formulation.

Effectiveness Assessment of a Modified Preservation Solution Containing Thyrotropin or Follitropin Based on Biochemical Analysis in Perfundates and Homogenates of Isolated Porcine Kidneys after Static Cold Storage.

In this paper, we assess the nephroprotective effects of thyrotropin and follitropin during ischaemia. The studies were performed in vitro in a model of isolated porcine kidneys stored in Biolasol (FZNP, Biochefa, Sosnowiec, Poland) and modified Biolasol (TSH: 1 µg/L; FSH 1 µg/L). We used the static cold storage method. The study was carried out based on 30 kidneys. The kidneys were placed in 500 mL of preservation solution chilled to 4 °C. The samples for biochemical tests were collected during the first kidney perfusion (after 2 h of storage) and during the second perfusion (after 48 h of storage). The results of ALT, AST, and LDH activities confirm the effectiveness of Biolasol + p-TSH in maintaining the structural integrity of renal cell membranes. Significantly reduced biochemical parameters of kidney function, i.e., creatinine and protein concentrations were also observed after 48 h storage. The protective effect of Biasol + p-TSH is most pronounced after 2 h of storage, suggesting a mild course of damage thereafter. A mild deterioration of renal function was observed after 48 h. The results of our analyses did not show any protective effect of Biolasol + p-FSH on the kidneys during ischaemia.

Biochemical Studies in Perfundates and Homogenates of Isolated Porcine Kidneys after Flushing with Zinc or Zinc-Prolactin Modified Preservation Solution Using a Static Cold Storage Technique.

Zinc is an effective anti-inflammatory and antioxidant trace element. The aim of this study was to analyse the protective effect of zinc and zinc-prolactin systems as additives of preservation solutions in the prevention of nephron damage caused during ischemia. The study used a model for storing isolated porcine kidneys in Biolasol®. The solution was modified with the addition of Zn at a dose of 1 µg/L and Zn: 1 µg/L with prolactin (PRL): 0.1 µg/L. After 2 h and 48 h of storage, the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, sodium, potassium, creatinine and total protein were determined. Zinc added to the Biolasol® composition at a dose of 1 µg/L showed minor effectiveness in the protection of nephrons. In turn, Zn2+ added to Biolasol + PRL (PRL: 0.1 µg/L) acted as a prolactin inhibitor. We do not recommend the addition of Zn(II) (1 µg/L) and Zn(II) (1 µg/L) + PRL (0.1 µg/L) to the Biolasol solution.

Preparation of Sterile Raw Material - Chicken Eggshells in the Process of their Transformation into Selected Calcium Salts.

BACKGROUND: The chicken eggshells and their subcrustal membranes are a valuable source of calcium, but they are not further processed but disposed of as waste from the food industry. Chicken eggshells have high content (>95%) of calcium carbonate. Some properties suggest that eggshells may be a promising alternative to the present calcium sources used in the pharmaceutical industry. METHODS: The effect of roasting chicken eggshells with a selected organic acid (citric or fumaric or lactic acid) on microbiological purity, including the presence of fungi and bacteria Salmonella spp., Staphylococcus aureus, Escherichia coli of obtained calcium salts, was investigated. In this study, chicken eggshells were subjected to chemical reactions with organic acids (citric, fumaric or lactic acid) at two different calcium-acid molar ratios (1:1 or 1:3) and the mixture was heat-treated for 1 or 3 hours at a temperature of 100°C or 120°C. RESULTS AND DISCUSSION: It was found that lactic acid was 100% effective against fungi, and the remaining citric and fumaric acids were -50% (regardless of the other examined conditions). The type of acid used has a significant effect on fungal growth inhibition (p<0.05). Fumaric acid and lactic acid will be nearly 100% effective against bacteria (100% fumaric acid and 97% lactic acid effectiveness), regardless of other factors. CONCLUSION: Lactic acid is the most effective against pathogenic flora - fungi and bacteria. The transformation of chicken eggshells into calcium lactate can provide us with sterile calcium salt, free of 100% fungi and 97% of all bacteria.

Therapeutic Potential of Selenium as a Component of Preservation Solutions for Kidney Transplantation.

Selenium has strong antioxidant properties and diverse effects on the immune system. The aim of the study was to analyse the protective effect of selenium as a component of a kidney preservation solution on the prevention of ischemia-reperfusion injury of nephrons. The solution was modified by the addition of Se (1 µg/L), prolactin (0.1 µg/L) and Se with prolactin (1 µg/L Se + 0.1 µg/L PRL). The study used a model for storing isolated porcine kidneys in Biolasol® (modified Biolasol®), which minimizes ischemia-reperfusion injury of grafts. The introduction of Se4+ ions at a dose of 1 µg/L into the Biolasol® preservation solution in the form of Na2SeO3 caused an increase in the activity/concentration of the analysed biochemical parameters: aspartate transaminase, alanine transaminase, urea and protein. This suggests an adverse effect of Se4+ on nephron function during ischemia-reperfusion. The best graft protection was obtained by using Biolasol® modified with the addition of selenium (IV) at a dose of 1 µg/L and prolactin at a concentration of 0.1 µg/L. We proposed the mechanism of prolactin action in the metabolic reduction of selenite (SO32-) during ischemia/reperfusion.

Influence of Concentration on Release and Permeation Process of Model Peptide Substance-Corticotropin-From Semisolid Formulations.

The transdermal route of administration of drug substances allows clinicians to obtain a therapeutic effect bypassing the gastrointestinal tract, where the active substance could be inactivated. The hormonal substance used in the study-corticotropin (ACTH)-shows systemic effects. Therefore, the study of the effect of the type of ointment base and drug concentration on the release rate and also permeation rate in in vivo simulated conditions may be a valuable source of information for clinical trials to effectively optimize corticotropin treatment. This goal was achieved by preparation ointment formulation selecting the appropriate ointment base and determining the effect of ACTH concentration on the release and permeation studies of the ACTH. Semi-solid preparations containing ACTH were prepared using Unguator CITO e/s. The release study of ACTH was tested using a modified USP apparatus 2 with Enhancer cells. The permeation study was conducted with vertical Franz cells. Rheograms of hydrogels were made with the use of a universal rotational rheometer. The dependence of the amount of released and permeated hormone on the ointment concentration was found. Based on the test of ACTH release from semi-solid formulations and evaluation of rheological parameters, it was found that glycerol ointment is the most favourable base for ACTH. The ACTH release and permeation process depends on both viscosity and ACTH concentration. The higher the hormone concentration, the higher the amount of released ACTH but it reduces the amount of ACTH penetrating through porcine skin.

Development and Study of Semi-Solid Preparations Containing the Model Substance Corticotropin (ACTH): Convenience Application in Neurodegenerative Diseases.

Corticotropin (ACTH, previously an adrenocorticotropic hormone) is used in the diagnosis and treatment of pituitary gland disorders, adrenal cortex disorders, and other diseases, including autoimmune polymyositis, systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, and ulcerative colitis. So far, the ointment dosage form containing ACTH for use on the skin is unknown. Therefore, it seems appropriate to develop a semi-solid formulation with corticotropin. Emulsion ointments were prepared using an Unguator based on the cream base Lekobaza® containing corticotropin in different concentrations, and then the physical and chemical parameters of the ointment formulations, such as pH, spreadability, rheological properties, and texture analysis, were evaluated. In addition, a USP apparatus 2 with enhancer cells was utilized to study the in vitro drug release characteristics of the selected formulations. All the ointments obtained were characterized by good spreadability and viscosity. An analysis of the ointment texture was performed and the dependence of the tested parameters on the ACTH content in the ointment was demonstrated. Examination of the structure of the ointment showed that a high concentration of ACTH increases the hardness and adhesiveness of the ointment. In turn, it adversely affects the cohesiveness and elasticity of the ointments tested. The results of the release study showed that ACTH is released the fastest from the formulation with the lowest concentration, while the slowest from the ointment with the highest concentration of ACTH.

The Effectiveness of Rinsing Organs With Selected Preservation Solutions Based on the Analysis of Their Physicochemical Parameters.

BACKGROUND: One of the key elements of successful transplantation is effectively rinsing off the blood and preserving organs under controlled hypothermia. The aim of the study was to analyze the physicochemical parameters of Biolasol (Biochefa, Sosnowiec, Poland) and histidine-tryptophan-ketoglutarate (HTK) (Custodiol) solutions, which are recommended for perfusion and preservation of abdominal parenchymal organs. METHODS: Biolasol and HTK solution were used for the study. The solutions were subjected to physicochemical analysis involving pH, density, osmolarity, viscosity, refractive index, zeta potential, hydrodynamic diameter, and rheological properties. Rheological parameters were associated with morphologic features of fluids. RESULTS: HTK and Biolasol are non-Newtonian systems with pseudoplastic properties and yield stress. The solutions begin to flow under shear stress greater than the ultimate stress. In addition, a nonlinear relationship of their viscosity as a function of velocity gradient (shear rate) was observed. CONCLUSIONS: The solutions reproduce blood properties, which leads to the conclusion about their effective filling of the vascular bed and high efficiency in organ rinsing.

Penetration of model hormones through the pericardium in simulated conditions in vivo.

The process of penetration of selected protein-peptide substances including insulin (INS), corticotropin (ACTH), prolactin (PRL) and albumin (reference protein) through the model membrane - pig pericardium was traced. These substances show a wide spectrum of therapeutic effects and diverse physicochemical properties (molecular weight, pI). The model substances penetrated the pericardium in simulated in vivo conditions from 1.0 mg / ml solutions. Based on the results obtained, pharmacokinetic parameters of the permeation process were determined - permeation rate (k), half-life (t50%) and their pharmaceutical availability (AUC [0-24 h]). All tested model substances penetrate the pericardium to different degrees. Within 24 h, they penetrate from 16.8% of albumin to 98.9% of insulin. Corticotropin penetrates 43.8% and PRL 34.2%. The highest availability is achieved with insulin, followed by ACTH, PRL and the lowest content of albumin. The results obtained suggest that the higher molecular weight of model protein-peptide substances, the lower the pericardial penetration (R2 = - 0.700) and availability (R2 = - 0.600), and the longer the half-life (R2 = 0.948).

Effect of Lutropin Concentration on the Efficiency of Isolated Porcine Kidney Storage in Modified Biolasol Solution.

BACKGROUND: Lutropin, a luteinizing hormone (LH), is a glycoprotein hormone secreted by gonadotropic cells of the anterior pituitary gland. LH receptors are a promising therapeutic target. The aim of the study was to analyze the protective effect of LH as a component of preservation solutions on the prevention of ischemia-reperfusion injury in isolated porcine kidneys. METHODS: The study used Biolasol solution (Biochefa, Sosnowiec, Poland) modified with the addition of ascorbic acid and LH at a concentration of 0.01 µg/L, 0.1 µg/L, and 1 µg/L. The study was carried out on 40 kidneys from 20 Polish large white pigs. Kidneys were divided into 4 groups, then washed out of the blood, and cooled by complete immersion in a 500-mL preservation solution at 4°C. The total storage time for grafts in the preservation solutions was 2 hours. After this time, the kidneys were perfused. Perfundates intended for biochemical analyses were collected from the renal vein after 0 minutes and after 30 minutes of perfusion. Another perfusion was performed after 24-hour renal storage. The activity of aspartate aminotransferase and alanine aminotransferase, as well as sodium, potassium, total protein, and urea concentrations was determined. RESULTS: LH as a component of preservation solutions affects the degree of ischemia-reperfusion injury of isolated porcine kidneys. CONCLUSIONS: The results obtained suggest that the efficacy of LH in improving ischemic kidney function is dependent on the dose used. The best graft protection was obtained using the lowest concentration of LH: 0.01 µg/L.

Effectiveness of absorption and passage through the small intestine, as a model of oral prolactin administration.

The process of absorption and permeation of PRL through the small intestine of 1-day-old piglet from the different compositions of solutions prepared for oral administration was investigated. This was achieved by determining the effect of hormone concentration (0.25 mg / ml or 0.5 mg / ml or 0.75 mg / ml), the concentration of stabilizing substances - trehalose (6 mg / ml or 12 mg / ml or 18 mg / ml) and mannitol (6 mg / ml or 12 mg / ml or 18 mg / ml) and the pH of the solution (2.5 or 3.0 or 3.5) on the degree of absorption and permeation of the PRL. The conditions for the absorption and penetration of PRL from solutions of various compositions for oral administration through the natural membrane (small intestine of the 1-day-old sucking piglet) in the in vivo conditions were simulated. The studies used an in vivo model in which the enzymatic profile in the body is not yet fully developed (no pepsin). It was found that in the studied range the absorption of PRL in the small intestine of the 1-day-old sucking piglet is significantly related to the concentration of the hormone and trehalose in the solution from which it is absorbed. In contrast, all factors studied (hormone concentration, trehalose and mannitol concentration, pH value of the solution) influence the process of penetration of the PRL in the studied range. It was also found that the hormone concentration significantly influences the rate of its absorption and permeation (the fastest occurs at a concentration of 0.5 mg/mL). The results suggest possibility of oral prolactin administration in order to ensure proper growth, development and increase the resistance and survival of sucking piglets.

The Effect of Modified Biolasol Solution on the Efficacy of Storing Isolated Porcine Kidneys.

Biolasol is a newly developed solution for storing the liver, pancreas, kidneys, and heart by simple hypothermia. It exhibits high efficacy in maintaining structural and functional integrity of the graft prior to its transplantation. The solution was modified by the addition of ascorbic acid (0.088g/l) and ascorbic acid with prolactin (1 µg/l PRL + 0.088g/l vitamin C). The effectiveness of the obtained solutions in the protection of nephrons of isolated porcine kidneys was assessed based on the analysis of the activity of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and LDH (lactate dehydrogenase) as well as lactate concentration determined in perfundates collected after 2 h (0' and 30' preservation) and 48 h (0' and 30' preservation) of graft storage. It has been found that the synergistic action of Biolasol components determines the integrity and stability of cell membranes, which in turn affects the proper functioning of the organ after transplantation. The addition of ascorbic acid and prolactin to Biolasol affects the maintenance of the normal cytoskeleton of the stored graft.

New Sources of Calcium (Chicken Eggshells, Chelates) - Preparation of Raw Material and Tablets.

BACKGROUND: There are many calcium supplements available in the market, especially those containing calcium in the form of carbonate, which unfortunately is not absorbed by the body to a sufficient degree. METHOD: Therefore, an attempt was made to prepare new sources of calcium, consuming the chicken eggshells as natural raw materials, which were used in preparation of tablets containing calcium carbonate and calcium citrate as well as tablets with calcium carbonate and calcium bisglycinate. The influence of raw material properties on the pharmaceutical availability of calcium from the obtained tablets was investigated. RESULTS: Based on the obtained calcium release profiles from the prepared tablets, it was found that the optimal source of calcium is a preparation containing calcium from chicken eggshells. It was found that both chicken eggshells and calcium bisglycinate (chelate) may be new, prospective sources of calcium. Calcium citrate prepared using eggshells as starting materials and bisglycinate is completely released within no more than 150 minutes. CONCLUSION: In turn, calcium carbonate added to calcium bisglycinate statistically significantly prolonges the release of calcium ions to 4 hours.

Tips for optimizing organ preservation solutions.

Organ injury during ischemia is one of the clinical problems of today's transplantation. It occurs during warm ischemia time (WIT) when the blood flow is cut off and during cold ischemia when a graft is chilled in situ until the circulation is restored to the recipient organism. Fast cooling of the organ slows down metabolism and activates intracellular enzymes, which minimizes the effects of warm ischemia. Unfortunately, hypothermia also results in inhibition of ATP synthesis, cell swelling and intracellular acidity. That is why research is continually being conducted to develop new fluids for rinsing and storing organs, as well as to optimize the composition of those that are already in use, which will allow for longer and more effective graft storage and restoration of their optimal functions after transplantation. This article provides current information on rinsing and storage fluids available on the global market. It also discusses tips for the fluid modifications with hormones and micronutrients.

Influence of plasma on the physical properties of ointments with quercetin.

Effects of two independent variables - the content of quercetin (0 or 1 or 1.5 or 5 %) and the content of plasma (0 or 2 or 4 or 6 %) - on the organoleptic properties and rheological parameters of model formulations prepared on an amphiphilic base were estimated. The consistency of all ointments was uniform, and the content of quercetin and plasma lay within the predefined range. Tested ointments are non-Newtonian systems. The content of quercetin and plasma was found to have a significant effect on the rheological properties of the ointments. An increase in the content of plasma in ointments was accompanied by a significant increase in their hardness, viscosity and shear stress and a reduction of their spreadability. The best rheological properties were shown by formulation F-3, containing 1.5 % of quercetin and 2 % of plasma.

Pharmacokinetics and Bioavailability of the GnRH Analogs in the Form of Solution and Zn2+-Suspension After Single Subcutaneous Injection in Female Rats.

BACKGROUND AND OBJECTIVES: Although many synthetic gonadoliberin analogs have been developed, only a few of them, including buserelin, were introduced into clinical practice. Dalarelin, which differs from buserelin by just one aminoacid in the position 6 (D-Ala), is not widely used so far. Gonadotropin-releasing hormone (GnRH) analogs are used to treat many different illnesses and are available in different forms like solution for injection, nasal spray, microspheres, etc. Unfortunately, none of the above drug formulations can release the hormones for 24 h. We assumed that classical suspension could solve this problem. METHODS: Two sets of experiments were performed. In the first one, buserelin and dalarelin were injected into mature female rats in two forms: suspension, in which the analogs are bounded by Zn2+ ions and solution. The pharmacokinetic parameters and bioavailability of the analogs were calculated, based on their concentration in the plasma measured by high-performance liquid chromatography method (HPLC). In the second experiment, the hormones in two different forms were injected into superovulated immature female rats and then the concentration of Luteinizing hormone (LH), Follicle-stimulating hormone (FSH) and 17ß-estradiol in the serum was measured by radioimmunological method. RESULTS: The Extent of Biological Availability (EBA), calculated on the base of AUC0-∞, showed that in the form of solution buserelin and dalarelin display, respectively, only 13 and 8 % of biological availability of their suspension counterparts. Comparing both analogs, the EBA of dalarelin was half (53 %) that of buserelin delivered in the form of solution and 83 % when they were delivered in the form of suspension. The injection of buserelin or dalarelin, in the form of solution or suspension, into superovulated female rats increased LH, FSH and estradiol concentration in the serum. However, after injection of the analogs in the form of suspension, the high concentration of LH and FSH in the serum persisted longer. CONCLUSION: Performed studies indicate that GnRH analogs in the form of suspension have higher bioavailability than their solution counterparts. It influences the effects of their action, especially in relation to LH and FSH.

Release of selected amino acids from zinc carriers.

The paper deals with the results of an investigation of the release of selected amino acids (histidine, tryptophan, tyrosine) from model suspensions prepared by co-precipitation with zinc chloride. It has been proven that the influence of the Zn(II)/amino acid molar ratio on dissolution profiles of the tested amino acids and dissolution half-life (t1/2) of histidine or tryptophan is significant. The amount of amino acid in the dispersed phase (supporting dose) is a determinant of the amino acid release profile. There is a minimal supporting dose (30.0 µmol of histidine or 17.4 µmol of tryptophan) that provides release of similar amounts of amino acid (4.1-4.6 µmol of histidine or 8.7-9.9 µmol of tryptophan) after the same time intervals. The tyrosine release profiles follow first order kinetics since the supporting dose (0.9-11.2 µmol) is limited by the tyrosine low solubility in water.

Distribution in Rats Internal Organs of Intraperitoneally Given 125I-Labeled Heptapeptide [2-8]-Leucopyrokinin ([2-8]-LPK), a Truncated Analog of Insect Neuropeptide Leucopyrokinin.

BACKGROUND: It was previously found that synthetic, insect-derived octapeptide leucopyrokinin (LPK) applied directly into the lateral brain ventricle induced a significant antinociceptive effect in rats. Its synthetic truncated analog heptapeptide [2-8]-leucopyrokinin displayed a stronger antinociceptive effect in comparison to native LPK. Moreover it was previously found a high accumulation of these both 125I-labeled peptides in adrenals, as well as in hypothalamus and in hippocampus of rats brain. OBJECTIVES: The aim of the present study was to assess the distribution of 125I-labeled [2-8]-leucopyrokinin in rats' internal organs an in several parts of the brain after peripheral - intraperitoneal (i.p.) administration. MATERIAL AND METHODS: The study was performed on male Wistar rats. A synthetic [2-8]-leucopyrokinin ([2-8]-LPK) was iodinated with Na125I. On the day of experiment a solution of 125I-[2-8]-LPK was i.p. injected and the next after 1 and 24 h animals were sacrificed by decapitation. Radioactivity levels in samples of parts of the brain and of internal organs were determined by counter Gamma Auto Count. RESULTS: A uniform, low accumulation 125I-[2-8]-LPK was found in evaluated samples of the brain and in internal organs. CONCLUSIONS: The results of the present study indicate a weak penetration into the brain and internal organs of intraperitoneally applied 125I-[2-8]-LPK in rats and correspond with previously determined weak biological effects of i.p. injected LPK and [2-8]-LPK.

Influence of selected factors on veins' permeability for albumin in vitro.

We present the results of a study on the influence of albumin and prolactin concentrations and intravascular fluid pH on vein permeability for albumin. Permeability conditions were simulated depending on albumin concentration, pH value and prolactin concentration. In research model an in vitro method was applied using natural membrane - porcine vena cava inferior. Vein permeability was in the 0.63% to 5.69% range. In control variant permeability was ~2.54% and increased ~2 fold at decreased albumin and PRL concentrations. At increased albumin concentration permeability was decreased 4-fold. Albumin concentration significantly influences albumin permeability.