We hypothesize that magnesium supplementation will not prevent, but may reduce the toxic effects of alloxan on pancreatic β cells. This may include the restoration of pancreatic glucose metabolism enzyme function back to the levels of the healthy control rats; decrease in LOOH comparing to alloxan-treated rats, reducing their levels back to the levels found in healthy control rats; activation of CAT and SOD, as protectors against ROS; increase in levels of G-SH and glutathione system enzymes, namely, G-Red and G-Per. Additionally, we hypothesize that magnesium supplementation may reduce fasting blood glucose levels in alloxan-induced diabetic rats due to the improved pancreatic function.

Twenty-five white Wistar rats (ca. 140 g each, same age) were used for the experiments. The animals were kept in cages under standard conditions (20 – 25 °C, 40 – 45% air relative humidity) with free access to compound feed, while water was strictly supplemented (20 mL daily). Compound feed composition remained unknown. All procedures with animals were carried out according to the “European Convention for the Protection of Vertebrate Animals for Experimental and Other Scientific Purposes” (Strasbourg, France, Council of Europe, March 18, 1986) and were approved by the local institutional ethical committee.

The rats were divided into five groups, 5 rats per group: intact control (group 1, C); induced diabetes (group 2, D); magnesium supplementation (100 mg Mg²⁺ kg ¹ weight, daily dose) with diabetes (group 3, Mg100-D); magnesium (250 mg.kg^-1) with diabetes (group 4, Mg250-D); magnesium (500 mg.kg^-1) with diabetes (group 5, Mg500-D). Day 0 was the date of dietary supplementation start, while alloxan induction of diabetes was performed on day 21, and all animals were sacrificed on day 30. Magnesium citrate (C₆H₆O₇Mg) was dissolved in tap water in the amount of 6.17, 15.44, and 30.88 mg per mL corresponding to 14, 35, or 70 mg Mg²⁺ in 20 mL water per animal, which relates to 100, 250, and 500 mg Mg²⁺ per kg of live weight. A single intraperitoneal injection of alloxan monohydrate ("Sinbias", Ukraine) in 0.85% physiological saline was performed on each rat from groups 2, 3, 4, and 5 (150 mg.kg^-1 of body weight) after 24-hours fasting on the 21^st day from the start of the experiment. All animals were sacrificed under ether anesthesia by decapitation on day 30, followed by removal of the pancreas within 2 minutes after the death. The removed pancreas was kept on ice for 5 to 6 minutes followed by bleeding through repeated perfusion with physiological saline (0.85% NaCl, 4 °C). The pancreas was chopped coarsely by scissors. One gram of chopped tissue was homogenized with 9.0 mL of 5 mM Tris-HCl buffer (1:10 dilution), pH 7.4, for 50 seconds at 0 °C (MRTU-421505-63, Ukraine). The homogenate was filtered through two layers of cheesecloth into a centrifuge tube followed by centrifugation (3,000 g, 10 minutes, 0 °C). The supernatant was used immediately for biochemical markers quantification and called “tissue sample” throughout the research paper.

All reagents used for the experiments, if not specified, were of laboratory grade or better.

Glucose determination was done on venous blood drawn from rat tails using glucose oxidase method (Gamma TM mini glucometer) after at least 6-hours fasting on days 21, 23, 25, 27, and day 30 (sacrifice day). The principle of the method is based on the reaction catalyzed by glucose oxidase:

(1) Glucose + O 2   → Glucolactone + H 2 O 2

using test strips and calibration strips supplied by the manufacturer.

LOOH determination is based on the interaction of ethanol extracts of lipid hydroperoxides with ammonium thiocyanate after precipitation of proteins with trichloroacetic acid (TCA). The original method developed by Wagner, Clever and Peters (1947), and later updated and modified by Mihaljević, Katušin-Ražem and Ražem (1996), was adapted. Briefly, 0.2 mL of tissue sample was placed in a centrifuge tube with 2.8 mL of ethanol (95%) and 0.05 mL of 50% TCA at 4 °C. The tube was tightly closed and shaken for 5 minutes, followed by centrifugation, resulting in protein precipitation (3,000 g, 10 minutes, 4 °C). 1.5 mL of the resulting supernatant was mixed with 1.2 mL of ethanol, 0.02 mL of concentrated HCl, and 0.03 mL of 1% Mohr’s salt, Fe(NH₄)₂(SO₄)₂, in 3% HCl. The mixture was shaken, followed by addition of 0.2 mL of 20% NH₄SCN. Optical absorbance (A₄₈₀) was measured after 10 minutes of incubation time (spectrophotometer UNICO S1205, λ = 480 nm). The control sample was prepared by tissue sample replacement with 0.2 mL of deionized water. LOOH in samples was calculated using the following equation:

(2) ΔA 480   LOOH .g -1   =   DF   x   ( A 480   exp   -   A 480   control )

The obtained results were expressed as the difference in optical absorbance between experimental and control samples (λ = 480 nm), adjusted to 1 g of pancreatic tissue (ΔA480 LOOH.g^-1) by dilution factor DF.

All reagents used for the experiments, if not specified, were of laboratory grade or better.

Protein concentration in the tissue sample was determined by the Lowry method using standardized kits (SIMCO Ltd, Ukraine) according to the manufacturer’s instructions. The method is based on the formation of non-ferrous products of the interaction of aromatic amino acids with the Folin-Ciocalteu reagent in combination with the biuret reaction to peptide bonds of the protein (Lowry et al., 1951). The results were expressed in mg of protein per mL of the tissue sample using spectrophotometer UNICO S1205 for absorbance measurements. The protein concentration of tissue samples from the pancreas of each rat was used to estimate enzyme activity per mg of protein in the subsequent analysis of SOD, CAT, G-Per, G-Red, G6PD, and LDH enzymatic activities in tissue samples.

The principle of the method for superoxide dismutase (EC 1.15.1.1) activity determination is based on the ability of the enzyme to compete with nitroblue tetrazolium for the superoxide anion radicals, which are formed as a result of aerobic interaction of NADH and phenazine methosulfate (Ponti et al., 1978). As a result of this reaction, nitroblue tetrazolium is reduced to formazan dye. In the presence of SOD, the percentage of nitroblue tetrazolium reduction is reduced. Enzyme activity was determined by the percentage of blockage of formazan dye formation (Durak et al., 1993). Briefly, 1.5 mL of incubation mixture (containing 37 mg EDTA-Na₂, 330 mg nitrotetrazolium blue, 55 mg phenazine methosulfate, and 0.3 mL 0.15 M phosphate buffer, pH 7.8) was mixed with 0.1 mL of a tissue sample and 0.1 mL of 1 mM NADH in Tris-EDTA buffer (pH 8.0) followed by 10 minutes incubation at room temperature. Optical absorbance was measured against water (UNICO S1205, λ = 540 nm). The control sample was prepared using distilled instead of a tissue sample. The enzyme activity was determined with the following equation:

(3) % blockage = 100 x (A c  -   A t )/A c

where A_c – optical absorbance of the control sample; A_t – optical absorbance of the test sample. Superoxide dismutase activity was expressed in relative units per 1 mg of protein (1 relative unit = 50% blocking).

The principle of the method for catalase (EC 1.11.1.6) activity determination is based on the ability of H₂O₂ to form a stable colored complex with molybdenum salts. According to Góth (1991), the color intensity of molybdenum peroxide compounds depends on the amount of H₂O₂ in solution; and therefore, on the activity of catalase in the sample. The catalase reaction was started by mixing 0.1 mL of a tissue sample with 1 mL of 0.05 M Tris-HCl buffer (pH 7.8) and 2 mL of 0.03% H₂O₂. Blank included 1 mL of 4% (NH₄)₂MoO₄ in 0.025 N H₂SO₄ and 2.0 mL H₂O₂. The reaction in the test sample was stopped after 10 minutes by adding 1 mL of 0.25 N H₂SO₄ and 1 mL of 4% (NH₄)₂MoO₄. To complete the blank, 1 mL of 0.25 N H₂SO₄ and 0.1 mL of tissue sample was added to the blank.

Samples were centrifuged for 5 minutes at 3,000 g. Optical absorbance was determined spectrophotometrically (UNICO S1205, λ = 410 nm). The catalase activity in μmol.min^-1.mg^-1 was calculated using the following equation:

(4) E a   =   ( ΔA   x   DF ) / ( ε   x   t   x   C   x   1 )

Where: ΔA – difference between the optical absorbance of blank and test samples; DF – dilution factor of the original tissue sample; Ԑ – molar extinction coefficient of the H₂O₂ complex with ammonium molybdate at λ = 410 nm, equal to 22200 M^-1.cm^-1; C – tissue sample protein concentration, mg.mL^-1; t – reaction time, 10 minutes, l – optical path, 1 cm.

All reagents used for the experiments, if not specified, were of laboratory grade or better.

The activity of glutathione peroxidase (EC 1.11.1.9) was measured through the oxidation rate of reduced glutathione in the presence of tertiary butyl hydroperoxide (Moin, 1996). Change in absorbance is due to the interaction between SH-groups and Ellman's reagent (0.01 M solution of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) in methanol) with the formation of a colored product, 5'-thio-2-nitrobenzoic acid. The amount of the latter is directly proportional to the number of -SH groups that reacted with Ellman’s reagent. Briefly, 0.83 mL of 4.8 mM reduced glutathione in 0.1 M Tris-HCl buffer (pH 8.5), containing 6 mM EDTA and 12 mM NaN₃, was added to 0.2 mL of the tissue sample. The mixture was incubated at 37 °C for 10 minutes. Following incubation, 0.07 mL of 20 mM tertiary butyl hydroperoxide was added and the mixture was incubated for an additional 5 minutes. The reaction was stopped by adding 0.2 mL of 20% trichloroacetic acid at 4 °C. A blank sample was prepared identically, but with distilled water instead of the tissue sample. The samples were centrifuged for 10 minutes at 3000 g. The supernatant (0.02 mL) was mixed with 2 mL of 0.1 M Tris-HCl buffer (pH 8.5) and 0.02 mL of Elman's reagent. After 5 minutes incubation, the samples were analyzed spectrophotometrically (UNICO S1205, λ = 412 nm). The G-Per result was expressed in μmol.min^-1.mg^-1 protein and calculated using the following equation:

(5) E a   =   ( ΔA   x   DF   x   C s ) / ( A s   x   t   x   C )

Where: ΔA – the difference between optical absorbance of the control and experimental samples; DF – dilution factor of the original tissue sample; A_s – absorbance of the standard G-SH solution; C_s – concentration of the standard G-SH solution; C – protein concentration in the tissue sample, mg.mL^-1; t – reaction time, 5 minutes.

The activity of glutathione reductase (EC 1.8.1.7) was determined spectrophotometrically as the rate of glutathione reduction in the presence of NADPH as suggested by Carlberg and Mannervik (1985). The reaction mixture contained 2 mL of 0.15 M phosphate buffer, pH 7.4; 0.2 mL of 10 mM EDTA; 0.5 mL of 7.5 mM oxidized glutathione, 0.1 mL of 2 mM NADPH; and 0.2 mL of the test sample. The enzyme activity was determined by measuring the reduction in NADPH content within 10 minutes at 37 °C (UNICO S1205, λ =340 nm). The activity of G-Red was expressed in μmol(NADPH).min^-1.mg^-1 protein and calculated using equation 4, where ΔA – absorbance difference between the start and end time of the reaction; DF – dilution factor of the tissue sample; Ԑ – molar extinction coefficient for NADPH, Ԑ = 6220 M^-1.cm^-1; t – reaction time, 10 minutes.

The content of reduced glutathione (G-SH) was determined by the level of 5'-thio-2-nitrobenzoic acid formation as a result of the interaction of the glutathione – SH groups with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) at λ = 412 nm as per method developed by Beutler, Duron and Kelly (1963). The following reagents were used: a precipitating reagent (glacial metaphosphoric acid – 6.68 g; EDTA – 0.80 g; sodium chloride – 120.0 g; distilled water to 400 mL); 0.3 M Na₂HPO₄ solution in distilled water; Elman's reagent (0.04% solution of 5.5-dithiobis-2-nitrobenzoic acid in 1% solution of trisodium citrate) (Rahman, Kode and Biswas, 2006).

Stage I. Sample: tissue sample – 2 mL, precipitating reagent – 3 mL; control: distilled water – 2 mL, precipitating reagent – 3 mL. The mixtures were kept at room temperature for 5 minutes followed by centrifugation at 3,500g, through which protein-free supernatant was obtained.

Stage II. Sample: supernatant from stage I – 2 mL, 0.3 M Na₂HPO₄ – 8 mL, Elman's reagent – 0.1 mL; Control: control supernatant from stage I – 2 mL; 0.3 M Na₂HPO₄ – 8 mL, Elman reagent– 0.1 mL. After 5 minutes of incubation, the absorbance measurement of the sample against control was performed (UNICO S1205, λ = 412 nm). The calculation of the amount of G-SH in the pancreas in mmoles per gram of tissue was estimated against G-SH standard solution with known concentration C_s using equation 6:

(6) N GSH  = (ΔA 412  x DF x C s )/A 412s

All reagents used for the experiments, if not specified, were of laboratory grade or better.

LDH and G6PD enzymatic activity were determined using spectrophotometric methods based on the coupled oxidation/reduction systems of nicotinamide coenzymes (UNICO S1205, 37 °C, NADH and NADPH molar extinction coefficient ε = 6220 at λ = 340 nm, t = 5 minutes of enzymatic reaction). The reaction was carried in 0.2 М tris-НСl buffer (рН 7.5) with a total volume of 3 mL, including 0.2 mL of the tissue sample. The final concentrations of the reaction components were as follows:

LDH – 1×10^-3 М sodium pyruvate, 5×10^-5 NADH, 3×10^-3 М MgCl₂ (Sevela and Tovarek, 1959).

G6PD – 1×10^-3 М glucose-6-phosphate, 5×10^-3 М MgCl₂; 0.5×10^-4 М NADP+ (Evans, 2009).

The enzymatic activity was expressed in nmole.min^-1.mg¹ protein and calculated using equation 4.

All reagents used for the experiments, if not specified, were of laboratory grade or better.

Two-factorial ANOVA was used to analyze the influence of treatment (D, Mg100-D, Mg250-D, Mg500-D) and time of experiment onset (day 21, day 25, day 27, and day 30), as well as their interaction (treatment x time), on the fasting glucose levels in rats. If the influence of factors or their interaction was significant (p <0.05), means were separated using the Fisher LSD procedure.

Pearson correlation coefficients were calculated between paired serum glucose levels of the control (C) and diabetes-induced rats (D) on day 30 and LOOH, SOD, CAT, G-Per, G-Red, G-SH, G6PD, and LDH levels in pancreas after animal sacrifice.

One-way ANOVA was used to analyze the influence of treatment (C, D, Mg100-D, Mg250-D, Mg500-D) on carbs metabolism enzyme activity, namely, G6PD and LDH; on lipid oxidative damage index, namely, LOOH level; on antioxidant enzymes activity, namely, SOD and CAT; and on glutathione system, namely, G-Per and G-Red activities and G-SH levels, in pancreatic tissue. If the influence of the factor was significant (p <0.05), means were separated using the Fisher LSD procedure.