Serum samples were used to perform the following measurements, through automatic validated methods and equipment (Hitachi 717 analyzer, Roche Diagnostics GMBH, Mannheim, Germany), as previously described [50 (link)]: postprandial glucose, triglycerides (TGs), total-cholesterol (Total-C), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels, as well as serum glutamic-oxaloacetic (GOT) and glutamic-pyruvic (GPT) transaminases concentrations. Hemoglobin A1c (HbA1c) levels were determined using the DCA 2000+ analyzer (Bayer Diagnostics, Barcelona, Spain), according to the manufacturer’ instructions. Serum insulin levels were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA) using commercially available kits for rat samples from Mercodia (Uppsala, Sweden). Insulin resistance was evaluated by the homeostatic model assessment of insulin resistance (HOMA-IR) index, which was calculated as previously described [51 (link)], using the following formula: HOMA-IR index = [fasting glucose (mmol/L) × fasting insulin (µU/L)]/22.5. High-sensitivity C-reactive protein (hs-CRP) was assayed by using a rat-specific Elisa kit (MBS764381 from Mybiosource, San Diego, CA, USA) according to the manufacturer’ instructions.
Dca 2000 analyzer
Manufactured by Bayer
Sourced in United States, Germany, Spain
The DCA 2000+ Analyzer is a compact and automated laboratory device used for the quantitative determination of glycated hemoglobin (HbA1c) in human blood samples. It utilizes principles of boronate affinity chromatography to perform this analysis.
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Lab products found in correlation
25 hydroxy chemiluminescent immunoassay, by DiaSorin (3 mentions)
Body composition analyzer, by Tanita (2 mentions)
Ultrasensitive nephelometry, by Siemens (2 mentions)
Glucose diagnostic reagents, by Merck Group (1 mentions)
Accu chek performa, by Roche (1 mentions)
Glucose cii test wako, by Fujifilm (1 mentions)
Lal assay, by Lonza (1 mentions)
Griess reagent system, by Promega (1 mentions)
Millennium chromatography software, by Waters Corporation (1 mentions)
7600 clinical analyzer, by Hitachi (1 mentions)
Quantitative sandwich enzyme immunoassay technique, by R&D Systems (1 mentions)
Diamat, by Bio-Rad (1 mentions)
Accu chek performa system, by Roche (1 mentions)
33 protocols using dca 2000 analyzer
1
Comprehensive Serum Biomarker Profiling
2
Monitoring Glycosylated Hemoglobin in T1D
3
Mouse HbA1c and Metabolic Measures
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Mouse immunoreactive HbA1c was determined using DCA 2000 analyzer (Bayer, Elkhart, IN). This system automatically measures both HbA1c and total hemoglobin in an approximately 5-μL blood sample [32 (link), 33 (link)]. Approximately 10–15 μL of blood was collected via a vein of mouse and measured HbA1c.
Blood collection was carried out after the 12 h in fasting state, on the final day of the second intervention (Exercise). To measure fasting blood glucose, samples were assessed via glucose diagnostic reagents (Sigma, St. Louis, MAK013). To evaluate insulin levels, blood was centrifuged for 15 min at 3 × 103g, and plasma was removed. The insulin levels were measured by the ELISA kit (mouse insulin Elisa, Alpco, 80-INSMS-E01). The homeostatic model assessment of insulin resistance( HOMA‐IR) as a scale of insulin resistance was determined by using the fasting blood glucose and insulin levels. The following equation was used to compute the HOMA‐IR value: Blood (plasma Insulin (μU/mL) × fasting Glucose (mg/dL) /405 [34 (link)].
To determine the concentration of triglyceride (TG), enzymatic methods were used in a calibrated biochemical analyzer (Hitachi 902 Automatic analyzer, Roche Diagnostics, USA) as follows. TG was measured by assessment of the produced H2O2 [35 (link)].
Blood collection was carried out after the 12 h in fasting state, on the final day of the second intervention (Exercise). To measure fasting blood glucose, samples were assessed via glucose diagnostic reagents (Sigma, St. Louis, MAK013). To evaluate insulin levels, blood was centrifuged for 15 min at 3 × 103g, and plasma was removed. The insulin levels were measured by the ELISA kit (mouse insulin Elisa, Alpco, 80-INSMS-E01). The homeostatic model assessment of insulin resistance
To determine the concentration of triglyceride (TG), enzymatic methods were used in a calibrated biochemical analyzer (Hitachi 902 Automatic analyzer, Roche Diagnostics, USA) as follows. TG was measured by assessment of the produced H2O2 [35 (link)].
4
Measuring HbA1c Using Immunoassay and DCA
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HbA1c was measured at each visit either by immunological in vitro assay (Tina-quant, Boebringer Mannheim Systems) or by a fingerstick blood sample with the DCA 2000+Analyzer (Bayer Inc., Tarrytown, NY, USA) following the manufacturer's guidelines. As we previously demonstrated the DCA 2000 levels correlated well with laboratory values (r = 0.88, p<0.001) with a mean difference of DCA 2000 and laboratory HbA1c values was 0.2% (95% confidence interval −0.1–0.6%) [15] (link).
5
Psychosocial and Glycemic Outcomes in Intervention
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The following self-report psychosocial measures were administered pre- and postintervention: the Perceived Stress Scale,26 (link) assessing an individual’s perception of life as stressful within the past month, adapted by increasing to 17 items and modifying wording to increase comprehension among urban Latino adolescents;3 (link) the Patient Health Questionnaire-9, a 9-item depressive symptom severity measure;27 (link) the General Well-Being Index, which has been used with participants as young as 14 years to measure psychological well-being;28 the Arizona Integrative Outcomes Scales,29 (link) single-item visual analogue scales measuring a person’s global state of physical, emotional, and spiritual well-being in the preceding 24 h and 30 days; the Satisfaction with Life Scale, a 5-item global life satisfaction measure. Glycemic control was assessed by measuring hemoglobin A1C using the DCA 2000 analyzer (Bayer Inc, Tarrytown, NY).
6
Tail Venous Blood Analysis in Mice
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Tail venous blood samples were obtained by a 1 mm incision on the tail tip on the day of surgery and on postoperative day 5, 10 and 21 or at a specific time. Blood glucose was then measured using the Antsense II Blood Glucose Analyzer (Bayer-Sankyo, Tokyo, Japan).33 Whole blood was collected on the days of sacrifice (online supplementary table 1 ) for hemoglobin A1c (HbA1c) assessment. Briefly, approximately 10 µL of blood was collected and 5 µL were added to a DCA 2000 analyzer (Bayer, Elkhart, Indiana, USA) which automatically measured Hb A1c as previously described.34 (link) Unpublished historical glucose and HbA1c data of a wildtype, non-diabetic Control group were used for comparison.
7
Evaluating Metabolic and Cognitive Outcomes
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Main outcome measures were: i) fasting blood glucose (FBG), 2-hour postprandial blood glucose (2hPG), and hemoglobin A1c (HbA1c) values before and six months after treatment; FBG and 2hPG were measured using Accu-Chek Performa blood glucose meter (Roche), while HbA1c was measured using DCA 2000 analyzer (Bayer); ii) MMSE and MoCA scores before and six months after treatment for cognitive evaluation.
Secondary outcome measures included C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and Aβ1–40 and Aβ1–42 values. Two tubes of venous blood (5 ml each) were collected from each patient at 8′clock in the morning before and six months after treatment. The blood samples were placed in sterile EDTA tubes and kept in a fridge at 4°C for 15 min. Afterward, samples were centrifuged at 1,500 × g at 4°C for 30 min to separate serum from plasma. The plasma was incubated with phosphate buffered saline containing 40 µl protease inhibitor at −80°C. Levels of CRP, TNF-α, and IL-6 in serum were measured using immunoturbidimetry, while values of Aβ1–40 and Aβ1–42 in plasma were measured using ELISA.
Secondary outcome measures included C-reactive protein (CRP), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and Aβ1–40 and Aβ1–42 values. Two tubes of venous blood (5 ml each) were collected from each patient at 8′clock in the morning before and six months after treatment. The blood samples were placed in sterile EDTA tubes and kept in a fridge at 4°C for 15 min. Afterward, samples were centrifuged at 1,500 × g at 4°C for 30 min to separate serum from plasma. The plasma was incubated with phosphate buffered saline containing 40 µl protease inhibitor at −80°C. Levels of CRP, TNF-α, and IL-6 in serum were measured using immunoturbidimetry, while values of Aβ1–40 and Aβ1–42 in plasma were measured using ELISA.
8
Antioxidant Capacity Measurement by TEAC Assay
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TEAC assay assesses the total radical scavenging capacity based on the ability of a compound to scavenge the stable ABTS•+ radical in 6 min. TEAC values of plasma samples were determined by a colorimetric assay based on the method described by Re et al [20 (link)]. The total antioxidant capacity was expressed as percentage of inhibition (PI), according to the equation: where AbsABTS•+ corresponds to the initial absorbance of diluted ABTS•+ and Abssample corresponds to the absorbance of the sample after 6 min of reaction. TEAC values were expressed as Trolox equivalents (TEs; mg Trolox/100g of sample) using the standard curve of Trolox. Final results were in µg of TE mL-1. Calibration curve ranged from 5 to 100 µg of TE mL-1.
Calibration standards were daily prepared, and all samples were determined in triplicate. For all the analysis, we have r ≥ 0.99.
HbA1c was determined with DCA 2000+ Analyzer (Bayer Inc., Tarrytown, NY, USA) immunoassay system (normal range: 3-6%).
Calibration standards were daily prepared, and all samples were determined in triplicate. For all the analysis, we have r ≥ 0.99.
HbA1c was determined with DCA 2000+ Analyzer (Bayer Inc., Tarrytown, NY, USA) immunoassay system (normal range: 3-6%).
9
Measuring Long-Term Glycemic Control
10
Measuring Long-Term Glycemic Control
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Hemoglobin A1C level, a measure of long-term glycemic control, was obtained by a trained research staff member using the DCA2000+ Analyzer from Bayer (Mikashawa, IN). Height was measured using a portable stadiometer at baseline. Weight was measured using a Tanita Body Composition Analyzer at each assessment (Tanita Corporation of America, Inc).
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