Age features of morphological changes of the hematotesticular barrier in experimental diabetes mellitus.

Background. Diabetes mellitus is an acute medical and social problem in all parts of the world due to the constant increase in the incidence of the disease, severe complications, disability and high mortality. Hyperglycemia leads to oxidative stress and increased processes of formation of active oxygen species, which in turn make a significant contribution to the development of male infertility. Objective. Therefore, the aim of our study was to establish morphological changes in the vessels of the hemomicrocirculatory flow and testicular sustentocytes of adult rats with experimental streptozotocin diabetes mellitus (SDM). Methods. The material for the study were the testicles of 20 sexually mature 6-month-old rats (weighing 150-180 g). SDM in animals of the experimental group was simulated by a single intraperitoneal injection of streptozotocin (dissolved in 0.1 M citrate buffer solution with a pH of 4.5) at a dose of 6 mg per 100 g of mass. An equivalent dose of 0.1 M citrate buffer was injected intraperitoneally to animals of the control group. Histological, electron microscopic, biochemical, morphometric and statistical research methods were used. Results. It was found that in the early stages of SDM (28th day) on the background of hyperglycemia in the hemomicrocirculatory flow of the testes there is a spasm of the vessels of the afferent link, which is confirmed by a decrease in the lumen of arterioles and an increase in their Wagenworth index. On the 70th day of SDM on the background of elevated levels of glucose and glycosylated hemoglobin in the links of the hemomicrocirculatory flow of the testis there are signs of diabetic microangiopathy, manifested by: hemorheological disorders in micro-hemo-vessels, (erythrocyte sludge, adhesion of erythrocytes and platelets, microclasmatosis), decrease in the capacity of arterioles and capillaries (increase in the Wagenworth index, respectively by 1.8 and 1.9 times), microclasmatosis, thickening and proliferation of the basement membrane of capillaries. Against the background of diabetic microangiopathy, there is a decrease in the number of sustentocytes by 0.01 mm2 of testicular parenchyma by 1.8 times, compared with control indicators, the area of their profile increases by 2.2 times (in all cases p<0.05). The nucleolar areas probably do not change, which leads to an increase in the nuclear-cytoplasmic ratio by 2.9 times (p<0.05). Such morphometric changes of sustentocytes are caused by development of vacuolar hydropic dystrophies in them, and an apoptosis that is confirmed by data of histo-ultrastructural studies. Changes in sustentocytes against the background of the development of diabetic microangiopathy lead to a violation of the hematotesticular barrier and to dystrophic changes in the spermatogenic epithelium of the testis. Conclusion. Thus, on the 70th day of SDM in the hemomicrocirculatory flow of the testis, the development of diabetic microangiopathy is observed, which leads to a violation of the hematotesticular barrier, and as a consequence, to a violation of spermatogenesis.

Nano Differential Scanning Fluorimetry-Based Thermal Stability Screening and Optimal Buffer Selection for Immunoglobulin G

Nano differential scanning fluorimetry (nanoDSF) is a high-throughput protein stability screening technique that simultaneously monitors protein unfolding and aggregation properties. The thermal stability of immunoglobulin G (IgG) was investigated in three different buffers (sodium acetate, sodium citrate, and sodium phosphate) ranging from pH 4 to 8. In all three buffers, the midpoint temperature of thermal unfolding (Tm) showed a tendency to increase as the pH increased, but the aggregation propensity was different depending on the buffer species. The best stability against aggregation was obtained in the sodium acetate buffers below pH 4.6. On the other hand, IgG in the sodium citrate buffer had higher aggregation and viscosity than in the sodium acetate buffer at the same pH. Difference of aggregation between acetate and citrate buffers at the same pH could be explained by a protein–protein interaction study, performed with dynamic light scattering, which suggested that intermolecular interaction is attractive in citrate buffer but repulsive in acetate buffer. In conclusion, this study indicates that the sodium acetate buffer at pH 4.6 is suitable for IgG formulation, and the nanoDSF method is a powerful tool for thermal stability screening and optimal buffer selection in antibody formulations.

Antidiabetic Potential of Silver/Chitosan/Ascorbic Acid Nanocomposites

Background: Diabetes mellitus is the most common health problem in the world. Silver nanoparticles (AgNPs) exposed great intrinsic anti-inflammatory, antibacterial, antiviral, and antifungal activities. Chitosan is an oligosaccharide biopolymer with a great ability to lower hyperglycemia, and ascorbic acid is a water-soluble vitamin with strong antioxidant activity. Objective: The present study aimed to estimate AgNPs/chitosan/ascorbic acid nanocomposite (Ag-NCs) anti-diabetic properties in streptozotocin-induced diabetic rats. Method: Eighteen male Wistar albino rats were divided into three main groups (6 rats/group); control, diabetic, and Ag-NCs groups. Control group: after a single dose of citrate buffer at PH 4.5 (0.1 mol/L, i.p), the rats orally received 1 ml distilled water daily for four weeks. The diabetic model was induced by a single dose of streptozotocin (60 mg/kg, i.p) for type 1 diabetes and the rats orally received 1 ml distilled water daily for four weeks. The diabetic group was treated orally with Ag-NCs (0.25 mg/Kg body weight) daily for four weeks. Results: AgNPs/chitosan/ascorbic acid nanocomposite group showed a reduction in the concentrations of glucose, NO, MDA, LDL, and the activities of AST, ALT, ALP, and GGT. At the same time, it caused a general increase in insulin, albumin, TB, TC, TG, HDL, CAT, SOD, and GSH levels. The histopathological investigation illustrated regeneration of damaged pancreatic beta cells and a clear improvement in the hepatic architecture. Conclusion: The suggested mechanism of action for Ag-NCs in decreasing diabetic complications in the liver involved two pathways; the hypoglycemic activity and the antioxidant role of AgNPs, chitosan, and ascorbic acid.

Anti-diuretic and anti-glycemic properties of Jatropha gossypiifolia L. leave extract on wistar rats

Background Jatropha gossypiifolia L. is a widespread plant in tropical and sub-tropical countries used in traditional medicine. This study investigated the anti-diuretic and anti-hyperglycemia activities of J. gossypiifolia leave extract on streptozotocin-induced diabetic rats. Methods The leaves was shade dried, pulverized and prepared into extract. 30, 50 and 100 mg/kg of the leaves extracts of J. gossypiifolia was subject to diuretics and hyperglycemic properties using established protocol of diuretic and diabetes test on the rat bladders emptied via mild compression in the pelvic region and gently pulling of their tails. 0.5 ml/kg normal saline, reference drug and the tested were administered with a single dose of the various drugs, and Streptozotocin (STZ) was freshly prepared in 0.1 M citrate buffer with pH 4.5 prior to induction, animals were fasted 24 h and single dose of 45 mg STZ per kg body weight was administered intraperitoneally. Urine and blood samples were isolated from rats and centrifuged for the determination of renal function test. Diuretic and antidiabetic indexes where evaluated using adopted method. Results This study showed that, graded doses of the extract significantly increased diuretic effect, specifically at 100 mg/kg increased diuretic index at 4.29 and urine volume 5.06 and 10 mg/kg Hydrochlorothiazide with 6.23 ml when compared untreated group (1.18 ml) (p < 0.0001). Also, it regulated renal function in homeostatic state. Graded doses at (30, 50 and 100 mg/kg) of the extract significantly reduced streptozotocine induced increased blood glucose level at day 14 (84.00, 60.67 and 42.00 IU/mL) when compared with 20 mg/kg glibenclamide and diabetics control (81.67 and 463.00 IU/mL) (p > 0.05). Also, the extract maintained a normal body mass indexes, biochemical and anatomical structure. Conclusion The effect associated with J. gossypiifolia potentiated its anti-diuretic and anti-hyperglycemic properties as early stated in the ethnomedicinal reports.

Critical Aspects in the Preparation of Extemporaneous Flecainide Acetate Oral Solution for Paediatrics

The availability of liquid oral preparations compounded by pharmacists is essential to meet paediatric needs which remain unanswered by the pharmaceutical industry. Unfortunately, compendial monographs are often not available and, in many cases, pre-formulation studies (e.g., compatibility with other excipients and solubility evaluations) are not performed in-depth, leading, in some rare cases, to the inadvertent administration of a toxic dose. In this study, the preparation of an oral liquid formulation for paediatric use, containing flecainide acetate at different strengths, was considered, taking into account the possible effects of conventionally used excipients. First, the optimal vehicle was selected based on a solubility study, evidencing some unexpected formations of precipitates. As a matter of fact, the buffers commonly used for oral solutions significantly reduced flecainide solubility, and the concomitant presence of citrate buffer and methylparaben even caused the formation of non-resuspendable crystals. Then, chemical, physical, and microbiological stability were assessed. Solutions at strengths of 10 and 20 mg/mL flecainide acetate were stable up to 8 weeks when compounded by using a 40% sucrose solution as a vehicle. Microbiological data showed that the use of methylparaben was not necessary over this time period.

Quantitative analysis of galactose using LDI-TOF MS based on a TiO2 nanowire chip

A novel method for quantifying galactose was developed to serve as a newborn screening test for galactosemia using laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry (MS) with a TiO2 nanowire chip. Herein, phosphate citrate buffer, serum, and dried blood spot (DBS) were employed for the quantitative analysis of galactose. To quantitatively analyze galactose, its reduction potential was used to oxidize o-phenylene diamine (OPD) into 2,3-diaminophenazine (DA), which were both detected using LDI-TOF MS with a TiO2 nanowire chip according to the concentration of galactose. The reproducibility and the interference of glucose were determined to demonstrate the applicability of this method. Moreover, mixtures of galactose, phenylalanine, and 17 α-OHP were analyzed to determine the interference induced by other biomarkers of metabolic disorders. The OPD oxidation of galactose was found to be selectively achieved under high-glucose conditions, similar to human blood, thereby showing good reproducibility. The intensities of the mass peaks of OPD and DA based on LDI-TOF MS with a TiO2 nanowire chip were linearly correlated in the galactose concentration range of 57.2–220.0 μg/mL (r2 = 0.999 and 0.950, respectively) for serum samples and 52.5–220.0 μg/mL (r2 = 0.993 and 0.985, respectively) for DBS after methanol precipitation/extraction. The enzyme immunoassay and LDI-TOF MS analysis results were statistically analyzed, and a mixture of phenylalanine, 17 α-OHP, and galactose was simultaneously investigated quantitatively at the cutoff level.

Analysis of Multiple Pesticides Residues in Market Samples of Okra and Associated Dietary Risk Assessment for Consumers

European Union Reference Laboratory method for Fruits and Vegetable (EURL-FV-2010-M1) for the quantification of pesticide residues was verified for the determination of multiple pesticides residues in okra. The targeted pesticides were extracted using acetonitrile with citrate buffer salts followed by cleanup with primary secondary amine (PSA) and analyzed on LC-MS/MS. The recoveries for all the targeted pesticides were within an acceptable range of 70.1 - 116.6% and precision in terms of RSD was 0.3 - 18.1%, respectively. The limit of quantification ranged from 0.002 mg/kg for carbofuran to 0.5 mg/kg for α-cypermethrin. The status of pesticide residues in okra (n= 21) available to consumers in the main markets of Pakistan has been determined by using this verified method. Sixty-two percent of the tested samples were contaminated out of which three samples were non-compliant with European Union Maximum residue limits (EU-MRL). The pesticides violating the EU-MRL were bifenthrin, thiamethoxam, and triazophos. For all the detected pesticides, estimated daily intake (EDI) ranged between 7.39×10−6 and 1.78×10−4 mg/kg of body weight while the values of Health Index (HI) fluctuated between 8.9×10−5 and 1.8×10−2. Although, residues of some pesticides were reported to be non-compliant with EU-MRLs, yet the risk posed by these pesticides to human health was insignificant.

Effect of Solvent on the Hydrodynamic Properties of Collagen

In this study, the effect of solvent on the hydrodynamic properties of collagen extracted from tail tendons of young rats was researched. Collagen was dissolved in various aqueous carboxylic acid solutions, including acetic acid (AA), acetic acid with the addition of sodium chloride (AA/NaCl), formic acid (FA), lactic acid (LA), citric acid (CA), and also citrate buffer at pH = 3.7 (CB). The properties of collagen solutions at a concentration of 0.45 mg/mL were characterized based on the viscometric method. The reduced viscosity, intrinsic viscosity, and Huggins coefficient of collagen solutions and effect of solvent, temperature, and UV irradiation on these properties were investigated. Collagen solutions in acetic acid, acetic acid/NaCl, and citrate buffer were irradiated with UV light up to 1 h, and the viscosity of collagen solutions was measured. It was found that the organic acids used as solvent affected viscosity behavior, denaturation temperature, and stability of collagen solutions. The lowest values of studied parameters were obtained for the collagen solutions in acetic acid with the addition of sodium chloride. Thus, the effect of various aqueous carboxylic acid solutions on collagen solutions properties and denaturation temperature can also be affected by the sodium chloride addition. The results of this research can be crucial for the preparation of collagen solutions for both cosmetic and biomedical applications.

Antioxidant and Esterases Profiling of diabetes-induced rat and tissues compartmentalisation.

Enormous complications associated with diabetes contribute to the therapeutic challenge confronting most of the world, including developing countries. This study was carried out to investigate diabetes mellitus on esterases and antioxidant enzymes in different tissues compartments of rats. Animals were divided into two groups of 10 animals each. The experimental group was confirmed diabetic by a single dose of streptozotocin injection (STZ, freshly dissolved in citrate buffer, pH 4.5, 50 mg/kg) intraperitoneally. In contrast, the control group was injected with citrate buffer only. Blood glucose and weight of the animals were monitored for 7 days. Blood, liver and brain were removed, and biochemical parameters determined spectrophotometrically. Diabetes produced various degrees of alterations in antioxidant defence mechanism and esterases activities that are compartment specific. Acetylcholinesterase (AChE) activity was inhibited to various extents. While AChE was inhibited to the tune of 39% in plasma, 33% in the brain and 30% in the liver, activation of the activity was observed in the red blood cell (RBC). The same trend of significant (p < 0.001) inhibition was observed with arylesterase in the plasma, brain and liver, and activation in the RBC. Diabetes induced significant (p<0.001) inhibition in catalase, Glutathione-S-transferase, and Superoxide dismutase in the brain and liver, respectively, compared to control more than the other compartments. However, activation was also observed in the RBC of these enzymes except for catalase and nitric oxide. In conclusion, distinct compartments effects of diabetes observed in this study could suggest a new approach for effective and safer therapeutics.