Elisa kit

The α-Al₂O₃ NPs (80 nm, 100% alpha, white, hydrophilic, purity 99%, SSA: > 15 m²/g, density 3.97 g/cm³ with rhombohedral crystallographic structure) and γ-Al₂O₃ NPs (20 nm, white, 99% purity, SSA: > 138 m²/g, density 3890 kg/m³, almost spherical morphology) were purchased from US Research Nanomaterials Inc., Houston, TX USA (CAS No 1344–28-1, USA). Additionally, all the ultra-pure chemicals used in this research were purchased from Merck Company. MDA (CAS No. ZB-MDA-A96A), GPX (CAS No. ZB-GPX-A96), T-SOD (CAS No. 706002), TAC (CAS No. ZB-TAC-A96) CAT (CAS No. 707002), iNOS (CAS No. ZB-10740C-R9648) were assessed using ELISA kits (Zellbio Germany) and cDNA kit (Thermo scientific, US, K1622). For immunohistochemical assays, the following reagents were used: TBS 1X solution (Sigma-T5912), PBS (Sigma-P4417), DAPI (Sigma- D9542) Normal Goat Serum (10%) (G9023-Sigma), Triton 3% (Sigma-T8787), Alexa Fluor 488 (Elabscience, China), CYP450: GTX15616, Secondary antibodies (mouse): orb688924. SYBR Green master mix (Addbio Co., Korea) was utilized, following specific standards for each marker. AST and ALT were assessed using related kits (Pars Azmun, Tehran, Iran).

All biochemical assays in the present study were carried out according to our previous study^{15 (link)}. In summary, the activities of Superoxide dismutase (SOD), Catalase activity (CAT), and glutathione peroxidase (GSH) in the homogenate of the gastrocnemius muscle were measured by the specific ELISA kits developed for rats according to their manuals instruction (ZellBio GmbH, Germany).

Plasma biochemical parameters, glucose, total protein (TP), albumin, globulin, high-density lipoproteins (HDL), low-density lipoproteins (LDL), cholesterol, triglycerides, lactate, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate transaminase (AST) and alanine aminotransferase (ALT) were analysed using commercial clinical investigation kits (Pars Azmoon Kit, Karaj, Iran). The antioxidant enzymes, including superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and malondialdehyde (MDA) were measured using ELISA kits, according to the kit protocol (ZellBio, GmbH, Frankfurt am Main, Germany). Cortisol levels in serum were measured using commercial kits (iCHROMA, Chuncheon-si, Republic of Korea) and the protocol provided with the kit.

GPX, SOD and CAT enzymes activity was measured in the liver homogenates using ELISA kits (ZellBio GmbH, Ulm, Germany) according to the kit instructions.

For this purpose, at the end of the fourth week, blood samples were taken from each patient. We evaluated the serum levels of IL-2, IL-6, IL-17, IFNγ, and TNFα with ELISA kits (ZellBio Co., Germany) according to the manufacturer’s instructions. Results were analyzed by an ELISA microplate reader (Bio Tek E1800, USA), and the concentrations were calculated.

A sample of 5 ml blood was collected from all participants after a 12 to 14 h fast, at baseline and at the end of week 15. These samples were centrifuged at 4000 rpm for 15 min. The samples of serum were separated into small aliquots and were frozen at − 80 °C. For Zn analysis, all tubes were washed by acid and rinsed with distilled water, then atomic absorption spectrometry (variant Chemthech Analytical 2000) was used to determine serum Zn concentration [39 (link), 40 ]. Serum concentration of high-sensitivity C-reactive protein (hs-CRP) was determined by enzyme-linked immunosorbent assay (ELISA) kits (Diagnostics Biochem Canada, Ontario, Canada) with an intra-assay coefficient of variation (CV) of 7.2%. Serum TNF-α was measured by ELISA kits (Diaclone, Besancon, France). Intra-assay CV for serum TNF-αwas 6.5%. Serum apelin concentration was assessed by ELISA kits (ZellBio GmbH, Ulm, Germany), with an intra-assay CV of 7.2%. Serum insulin was determined by ELISA kits (Monobind, USA), with an intra-assay CV of 7.4%. Serum glucosewas measured by commercial kits (Pars Azemoon, Tehran, Iran) with the aid of a Selectra 2 Autoanalyzer (Vital Scientific, Spankeren, The Netherlands). Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) was determined using the following equation: $$\text{HOMA-IR}=\left[\text{Fasting serum glucose}\left(\text{mg}/\text{dL}\right)\times \text{Insulin}\left(\mathrm{\mu }\text{U}/\text{L}\right)\right]/405$$