Diabetes was induced in rats by intraperitoneal administration of alloxan. Rats were divided into two groups (eight rats each): the control group (C) and the alloxan-induced diabetic group. Prior to the experiments, rats (220–240 g) were fed standard rodent food for one week for acclimation to the laboratory conditions. The acclimated rats were fasted for 12 h with free access to water, and then injected with 0.5% alloxan dissolved in 10 mM sodium citrate (pH 4.5) at a dose of 150 mg/kg. Under the same conditions, C rats were injected with a saline solution. After 5 days, blood glucose was measured using a «Saterlit» glucometer. After 5 days, the animals were fasted, anesthetized with halothane, and dissected .
The experimental protocol corresponded to the conditions of the European Communities Council Directive (2010/63/ UE) and was approved by the Ethics Committee of Yerevan State Medical University after Mkhitar Heratsi (IRB Approval N4, November 15, 2018).
Isolation and purification of О2
−-producing associate NLP- Nox from rat`s SI
NLP – Nox associates from rat SI were isolated and purified using a universal method , which uses human ferrihemoglobin (Hb) for the release of Nox and NLP-Nox from SI . In particular, a water homogenate of SI (up to 6 g) was incubated for 1.5 h at pH 9,5 and 37 °C in the presence of 50 mM human Hb. The pH of the supernatant was adjusted to 4 after centrifugation at 5000 g for 10 min. The precipitate of the NLP-Nox fraction was soluble in water at pH9.5. After centrifugation, the supernatant was subjected to ion-exchange chromatography using cellulose DE-52 at pH 9.5. The Nox-NLP associate was eluted free because it was not absorbed by the column. The Nox fraction was eluted with 0.2 M potassium phosphate buffer (PPB) at pH 7.4. Following the concentration of the Nox and NLP-Nox fractions, gel filtration was performed on a separate Sephadex G-100 column at pH 9.5. The fractions of NLP-Nox and Nox eluted with a symmetrical elution diagram were collected and vacuum lyophilized after deionization of the Nox and NLP-Nox associates. They were weighed and stored in closed containers under a nitrogen atmosphere at -10oC.
Electrophoresis of the Nox-NLP associates was performed on a 10% PAAG (polyacrylamide gel) for proteins of acidic or basic characteristics.
Determination of NADPH in the composition of SI NLP-Nox
The presence of NADPH in the composition of SI NLP-Nox was determined using a spectrofluorimetric method that measured the fluorescence intensities in comparative units (F) at 430 nm with excitation at 370 nm .
Determination of the lipid component in the SI NLP-Nox composition
The lipid component of SI NLP-Nox was determined by measuring the lipid peroxidation product, malondialdehyde (MDA) .
Determination of the Nox in the NLP-Nox composition of SI
The Nox in the composition of NLP-Nox associate from SI was determined using the optical absorbance characteristic for Nox at 558, 525, and 418 nm in the reduced states by sodium dithionite.
Determination of the stationary concentration of О2
−produced by SI NLP-Nox associate
The stationary concentration of О2− produced by NLP-Nox associate from SI was determined using the adrenaline method. The maximal optical absorbance of adrenochrome (at 500 nm), which is formed during the oxidation of adrenaline by produced О2− , was determined. At the same, the stationary concentration (M) of produced О2− is equal to the concentration of formed adrenochrome, with a molar extinction (E) of up to 750 M-1 cm-1. The stationary concentration (M) of О2− produced by this associate NLP-Nox was determined in the homogeneous phase (in solution) and gas phase by determining the value of A500/E. The optical absorbance of adrenochrome, which is formed during the oxidation of adrenaline by air oxygen, was used as a control.
The content of the lipid component (malondialdehyde) in NLP-Nox associate from SI in AD rats was 22.4% higher than that in C rats.
The specific content of NLP-Nox was determined by weighing it after deionization and vacuum lyophilization and was expressed in mg per 1 g SI (mg/g).
The cellulose of DE-52 («Whatman», England), Sephadex G-100 («Pharmacia», Sweden), adrenaline («Sigma», USA), spectrophotometer «Cary 60» (USA), spectrofluorometer «Perkin-Elmer», (USA), centrifuge K-70D and K-24 «Janetzki» (Germany) were used during the investigation.
Statistical analysis of the results using the variational statistics method of Student-Fisher was performed to determine the validation criterion (M ± m).