Blood samples were drawn after a 12-h fast, before surgery. Serum was separated and immediately frozen at –80 °C. Serum biochemical parameters were measured in duplicate. Serum glucose, cholesterol, HDL cholesterol and triglycerides were measured by standard enzymatic methods (Randox Laboratories Ltd, Antrim, UK). Insulin was measured with an immunoradiometric assay (BioSource International, Camarillo, CA, USA). The HOMA-IR was calculated from fasting insulin and glucose using the following equation: HOMA-IR=fasting insulin (μIU/ml) × fasting glucose (mol/l)/22.5. Serum survivin was measured by a sandwich ELISA (R&D Systems, Minneapolis, MN, USA). The assay sensitivity was 1.58–9.96 pg/ml and the intra- and inter-assay co-efficients of variance (CVs) were less than 5.5 and 9.5%, respectively.62 (link) Serum leptin was measured by ELISA (R&D Systems). The minimum detectable concentration of human leptin is typically less than 7.8 pg/ml and the intra- and inter-assay CVs were less than 3.3 and 5.4%, respectively.
Immunoradiometric assay
Manufactured by Thermo Fisher Scientific
Sourced in United States, Belgium
Immunoradiometric assay is a type of radioimmunological technique used for the quantitative measurement of specific analytes, such as hormones, proteins, or other biomolecules, in a sample. It employs radioactive isotope-labeled antibodies or antigens to detect and quantify the target analyte.
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Lab products found in correlation
Dimension autoanalyzer, by Siemens (3 mentions)
Elisa kit, by Mediagnost (2 mentions)
Hexokinase activity assay, by Abcam (2 mentions)
Triglycerides glycerol blanked, by Roche (2 mentions)
Dimension autoanalyzer, by Roche (2 mentions)
Ldl cholesterol plus 2nd generation, by Roche (2 mentions)
Cholesterol gen 2, by Roche (2 mentions)
747 automatic analyzer, by Hitachi (2 mentions)
Irisin elisa kit ek 067 52, by Phoenix Pharmaceuticals (2 mentions)
Elisa, by R&D Systems (1 mentions)
Sandwich elisa, by R&D Systems (1 mentions)
Aeroset system, by Abbott (1 mentions)
Enzyme linked immunosorbent assay kit, by Thermo Fisher Scientific (1 mentions)
Adiva 1800, by Siemens (1 mentions)
D 100, by Bio-Rad (1 mentions)
1470 wizard gamma counter, by PerkinElmer (1 mentions)
7600 automated analyzer, by Hitachi (1 mentions)
25 hydroxyvitamin d 125i radioimmunoassay kit, by DiaSorin (1 mentions)
Elisa kit, by Immundiagnostik (1 mentions)
Leptin elisa kit, by Mediagnost (1 mentions)
26 protocols using immunoradiometric assay
1
Serum Biomarkers in Surgical Patients
2
Fasting Biochemical Parameters and Hormones
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After a 12 h fast, baseline laboratory parameters, including serum glucose, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol, triglycerides (TGs), high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol, were measured using the auto analyzer system (Aeroset System Abbott, Abbott Laboratories, Diagnostic Division, Chicago, IL, USA) [37 (link)].
Further, once plasma samples were collected following previous procedures described elsewhere [21 (link)], leptin levels were measured by means of an enzyme immunoassay (ELISA) kit (cat. No. EH0216; FineTest, Wuhan, China), insulin was analyzed by an immunoradiometric assay (BioSource International, Camarillo, CA, USA), and ghrelin was determined by an enzyme-linked immunosorbent assay kit (cat. No. EH0355; FineTest, Wuhan, China). The methods of analysis and concentration measurements followed established procedures [21 (link)].
Further, once plasma samples were collected following previous procedures described elsewhere [21 (link)], leptin levels were measured by means of an enzyme immunoassay (ELISA) kit (cat. No. EH0216; FineTest, Wuhan, China), insulin was analyzed by an immunoradiometric assay (BioSource International, Camarillo, CA, USA), and ghrelin was determined by an enzyme-linked immunosorbent assay kit (cat. No. EH0355; FineTest, Wuhan, China). The methods of analysis and concentration measurements followed established procedures [21 (link)].
3
Fasting Blood Biochemical Analysis
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Blood samples were drawn after a 12-h fast. Serum/plasma was separated and immediately frozen at −80 °C. Serum biochemical parameters were measured in duplicate. Serum glucose, cholesterol, high density lipoprotein (HDL) cholesterol and triglycerides were measured by standard enzymatic methods (Randox Laboratories Ltd, Antrim, UK). Insulin was measured with an immunoradiometric assay (BioSource International, Camarillo, CA).
4
Metabolic Biomarkers in Fasted Subjects
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Blood samples from all subjects were collected before surgery and after a 12-h fast. The serum was separated and immediately frozen at −80 °C. Serum biochemical variables were measured in duplicate. Serum glucose, cholesterol and triglycerides (Randox Laboratories Ltd., Antrium, UK) were measured by standard enzymatic methods. Adiponectin levels were measured by enzyme immunoassay (ELISA) kits (DRG Diagnostics, Marburg, Germany). Leptin levels were measured by ELISA kit from Mediagnost (Reutlingen, Germany). The insulin was analyzed by an immunoradiometric assay (BioSource International, Camarillo, CA, USA). HOMA-IR was calculated with the following equation: HOMA-IR = fasting insulin (µIU/mL) × fasting glucose (mmol/L)/22.5 [22 (link)].
5
Fasting Biomarker Measurement Protocol
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Venous blood samples were drawn from each patient after fasting for 8 h or overnight. Blood samples were centrifuged to obtain plasma that was stored at − 80 °C. Plasma glucose was measured using the glucose oxidase method, and serum insulin levels were measured using an immunoradiometric assay (Biosource, Nivelles, Belgium). Total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol levels were determined using enzymatic methods with standard biochemical procedures on a BM Hitachi automated clinical chemistry analyzer (Hitachi, Tokyo, Japan).
6
Metabolic Profile Characterization of Participants
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Blood samples from all participants were obtained after an overnight fast and before surgery. Serum was separated by centrifugation for 15 min at 4000 rpm at 4 °C and frozen at −80 °C. Serum Glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL), and alkaline phosphatase measurements were carried out using a Dimension Autoanalyzer (Dade Behring Inc., Deerfield, IL, USA). The insulin levels were performed using an immunoradiometric assay (BioSource International, Camarillo, CA, USA). Calculated values for low-density lipoprotein cholesterol (LDL) was obtained by the Friedewald formula [54 (link)]. The HOMA-IR was obtained by the following equation: HOMA-IR = fasting insulin (μIU/mL) × fasting glucose (mmol/L)/22.5 [55 (link)].
7
Evaluation of Metabolic and Safety Outcomes
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Glucose levels were determined using the standard glucose oxidase method (Beckman Coulter Glucose Analyzer; Beckman Coulter Inc., CA, USA). Insulin was determined by immunoradiometric assay (Biosource Europe S.A., Nivelles, Belgium). Within and between assays, coefficients of variation for insulin were 3.6 and 3.8%. HbA1 was assessed by high-performance liquid chromatography (D-100; Bio-Rad Laboratories). Within and between assays, coefficients of variation for HbA1c were 1.67 and 2.27%. Lipids were determined using Adiva 1800, Siemens analyzer. Insulin resistance (IR) was calculated by the homeostasis model assessment for IR (HOMAIR): fasting serum insulin (mU/L) × fasting plasma glucose (mmol/L)/22.5 (18 (link)). Impaired glucose tolerance (IGT) was identified by 2 h glucose levels between 7.8 and 11.0 mmol/L, as defined by the American Diabetes Association criteria (19 (link)).
Comorbid conditions included self-reported heart condition, diabetes, cancer, liver conditions, kidney conditions, prostate disease and thyroid disorders. The self-reported history was checked and completed by available medical records.
Safety parameters (complete blood count, PSA, markers of hepatic and renal functions and serum electrolytes) were assessed before and after 16 weeks of study treatment. All men were instructed to report any side effects during the treatment.
Comorbid conditions included self-reported heart condition, diabetes, cancer, liver conditions, kidney conditions, prostate disease and thyroid disorders. The self-reported history was checked and completed by available medical records.
Safety parameters (complete blood count, PSA, markers of hepatic and renal functions and serum electrolytes) were assessed before and after 16 weeks of study treatment. All men were instructed to report any side effects during the treatment.
8
Measuring Metabolic Biomarkers in Blood
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Blood samples were obtained by venipuncture, refrigerated immediately, and transported to the central testing institute. Fasting plasma glucose and serum lipids (total cholesterol, high-density lipoprotein cholesterol, and triglycerides) were measured enzymatically with the Hitachi automated analyzer 7600 (Hitachi, Tokyo, Japan), and fasting plasma insulin was determined using a 1470 WIZARD gamma-Counter (Perkin-Elmer, Turku, Finland) and an immunoradiometric assay (Biosource, Fleurus, Belgium). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as a marker of insulin resistance, as follows:
The 25-hydroxyvitamin D concentration was measured by radioimmunoassay using a 1470 WIZARD gamma-Counter (Perkin-Elmer) and a 25-hydroxyvitamin D 125I radioimmunoassay kit (DiaSorin, Stillwater, MN, USA). At serum 25-hydroxyvitamin D levels of 8.6, 22.8, 33.1, and 49.1 ng/mL, the inter-assay coefficients of variation (CV) were 11.7%, 10.5%, 8.6%, and 12.5%, respectively, and the intra-assay CVs were 9.4%, 8.2%, 9.1%, and 11.0%, respectively.
The 25-hydroxyvitamin D concentration was measured by radioimmunoassay using a 1470 WIZARD gamma-Counter (Perkin-Elmer) and a 25-hydroxyvitamin D 125I radioimmunoassay kit (DiaSorin, Stillwater, MN, USA). At serum 25-hydroxyvitamin D levels of 8.6, 22.8, 33.1, and 49.1 ng/mL, the inter-assay coefficients of variation (CV) were 11.7%, 10.5%, 8.6%, and 12.5%, respectively, and the intra-assay CVs were 9.4%, 8.2%, 9.1%, and 11.0%, respectively.
9
Metabolic Biomarker Measurement Protocol
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Before surgery, and after an overnight fast, blood samples were obtained from the antecubital vein and placed in vacutainer tubes (BD vacutainer™). The serum was separated by centrifugation for 15 min at 4000 rpm and immediately frozen at −80 °C until analysis. Insulin was analyzed by an immunoradiometric assay (BioSource International, Camarillo, CA, USA) in a Beckman Coulter (Fullerton, CA, USA), showing 0.3% cross-reaction with proinsulin. The intra-assay and inter-assay CV was 1.9% and 6.3%, respectively. Serum glucose, cholesterol, triglycerides, HDL cholesterol (HDL-C), and C-reactive protein (CRP) were measured in a Dimension autoanalyzer (Dade Behring Inc., Deerfield, IL, USA) by enzymatic methods (Randox Laboratories Ltd., Barcelona, Spain). Glucose intra-assay CV was 7.5% while inter-assay CV was 13.5%. LDL cholesterol (LDL-C) was calculated using the Friedewald equation. Insulin was quantified by radioimmunoassay supplied by BioSource International Inc., Camarillo, CA, USA. HOMA-IR was calculated with the following equation: HOMA-IR = fasting insulin (µIU/mL) × fasting glucose (mmol/L)/22.5. Vitamind D or 25(OH)D levels were determined by enzyme immunoassay (ELISA) kits (Immundiagnostik AG, Bensheim, Germany), deficiency was considered if levels were <20 ng/mL (50 nmol/L). The inter-assay CV was 5.6% and the intra-assay CV was 7.3% [37 (link)].
10
Serum Biochemical Markers in Morbid Obesity
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The blood samples from all the subjects before surgery were collected after a 12-h fast. The serum was separated and immediately frozen at −80 °C. The serum biochemical variables were measured in duplicate. Serum glucose, cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides, and FFAs were measured by standard enzymatic methods (Randox Laboratories Ltd., Antrium, UK). The adiponectin levels were measured by an enzyme immunoassay Adiponectin ELISA kit (DRG Diagnostics, Marburg, Germany). The leptin levels were measured by a Leptin ELISA kit (Mediagnost, Reutlingen, Germany). The insulin was analyzed by an immunoradiometric assay (BioSource International, Camarillo, CA, USA). The homeostasis model assessment of insulin resistance index (HOMA-IR) was calculated with the following equation: HOMA-IR = fasting insulin (μIU/mL) × fasting glucose (mg/dL)/405 [28 (link)]. The morbidly obese subjects were also divided in two groups as follows: those with low HOMA-IR (<4.7) and those with high HOMA-IR (>8).
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