Blood and urine samples were collected from clinically healthy and diabetic dogs on day 0 for the baseline data. The blood and urine samples from the diabetic dogs were reassessed every 30 days for 90 or 180 days, depending on the treatment protocol. Blood samples (3–5 mL) were collected from the cephalic or saphenous vein to evaluate the clinical parameters and the biomarkers for inflammation and oxidative stress. The clinical parameters, which included complete blood count (CBC) and the levels of glucose, fructosamine, alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine, were analyzed every 30 days during routine health check-up. IL-6 and TNF-α were selected as the inflammatory biomarkers, whereas SOD and MDA were used as the oxidative stress biomarkers. The biomarkers were evaluated on days 0 and 90 or 180, depending on the treatment protocol.
One drop of the blood sample was used to test the glucose level using the AlphaTRAK glucometer (Zoetis, Parsippany, NJ, United State). The blood samples were then divided into three parts and used for further experiments. The ethylenediaminetetraacetic acid (EDTA) blood samples were stored at 4°C, and the CBCs were measured using an animal blood counter (Horiba Medical, Montpellier, France) within 4 h. The second part was centrifuged at 3500 rpm for 5 min within an hour after blood collection; the resultant plasma was used to measure the ALT, ALP, BUN, and creatinine levels using an automatic analyzer (Chema diagnostica, Monsano AN, Italy), and the serum was collected in a sterile microcentrifuge tube and stored at −80°C to measure the IL-6, TNF-α, SOD, and MDA levels. The third part was collected in plain tubes and sent to a commercial laboratory within 24 h to measure the fructosamine level.
The urine sample was collected by cystocentesis and centrifuged at 2000 rpm for 3 min (Hettich Lab Technology, Tuttlingen, Germany). The physical and chemical properties (color, clarity, specific gravity, pH, protein, glucose, ketone, and bilirubin levels, and erythrocyte counts) of the urine supernatants were tested using the dipstick test (Roche Diagnostics, Indianapolis, IN, United State). The urine sediments were evaluated under a light microscope (ZEISS, Jena, Germany). The white blood cell counts (high-power field: HPF), red blood cell counts (HPF), and presence of amorphous crystals, mucous, bacteria, epithelium cells (HPF), cast (low-power field: LPF), and crystals were determined.
One drop of the blood sample was used to test the glucose level using the AlphaTRAK glucometer (Zoetis, Parsippany, NJ, United State). The blood samples were then divided into three parts and used for further experiments. The ethylenediaminetetraacetic acid (EDTA) blood samples were stored at 4°C, and the CBCs were measured using an animal blood counter (Horiba Medical, Montpellier, France) within 4 h. The second part was centrifuged at 3500 rpm for 5 min within an hour after blood collection; the resultant plasma was used to measure the ALT, ALP, BUN, and creatinine levels using an automatic analyzer (Chema diagnostica, Monsano AN, Italy), and the serum was collected in a sterile microcentrifuge tube and stored at −80°C to measure the IL-6, TNF-α, SOD, and MDA levels. The third part was collected in plain tubes and sent to a commercial laboratory within 24 h to measure the fructosamine level.
The urine sample was collected by cystocentesis and centrifuged at 2000 rpm for 3 min (Hettich Lab Technology, Tuttlingen, Germany). The physical and chemical properties (color, clarity, specific gravity, pH, protein, glucose, ketone, and bilirubin levels, and erythrocyte counts) of the urine supernatants were tested using the dipstick test (Roche Diagnostics, Indianapolis, IN, United State). The urine sediments were evaluated under a light microscope (ZEISS, Jena, Germany). The white blood cell counts (high-power field: HPF), red blood cell counts (HPF), and presence of amorphous crystals, mucous, bacteria, epithelium cells (HPF), cast (low-power field: LPF), and crystals were determined.
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