The biochemical analysis was performed to measure the serum level of triglycerides (TG), total cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), serum glutamic oxaloacetic transaminase (SGOT/AST), serum glutamic pyruvic transaminase (SGPT/ALT), alkaline phosphatase (ALP), urea, creatinine, and total protein (TP). Analytical tests were performed via UV–vis spectrophotometer using Konelab 20XTi (Thermo Fisher Scientific, USA).
Konelab 20xti
Manufactured by Thermo Fisher Scientific
Sourced in United States, Finland
The Konelab 20XTi is a benchtop clinical chemistry analyzer designed for in vitro diagnostic testing. It is capable of performing a wide range of routine and specialized biochemical assays, including colorimetric, enzymatic, and ion-selective electrode measurements. The Konelab 20XTi is intended for use in clinical laboratories and other healthcare settings.
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21 protocols using konelab 20xti
1
Comprehensive Biochemical Analysis Panel
2
Lipid Profile Measurement Protocol
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Venous blood samples were collected before the preparatory RT period and every four weeks thereafter. Venous blood samples were drawn after 12 h of fasting to obtain concentrations of total cholesterol, LDL, HDL and triglycerides. Subjects were asked to rest for at least 8 h during the preceding night and were required to refrain from strenuous physical activity for at least 48 h. Blood samples were taken from the antecubital vein into serum tubes (Venosafe; Terumo Medical Co., Leuven, Hanau, Belgium) using standard laboratory procedures. Blood samples were stored in room temperature for 10 min, after which they were centrifuged at 3500 rpm for 10 minutes (Megafure 1.0 R Heraeus; DJB Lab Care, Germany) and the serum obtained was immediately analyzed by spectrophotometry (Konelab 20XTi; Thermo Fisher Scientific, Vantaa, Finland). LDL concentration was estimated using the Friedewald [33 (link)] equation: LDL = total cholesterol - HDL - (triglycerides/2.2).
3
Automated Blood Cell Analysis
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Estimated total white blood cell counts and differential counts were routinely done.9 Thrombocyte numbers were considered normal if more than two thrombocytes were seen per field at 1000× magnification. Polychromasia was scored as 0 (no polychromatophilic cells) or 1+ (1–5% polychromatophilic cells). The heparinized blood was centrifuged and plasma was removed and frozen at −20°C. Plasma biochemical analytes were determined using a Konelab 20xti (Thermo Fisher Scientific, NSW, Australia).
4
Oxidative Stress and Muscle Damage
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Fasting blood samples will be obtained via venepuncture from the median cubital vein, while urinary measures will be determined from a 24 hours urine collection. Plasma samples will be prepared in accordance to Barden et al53 (link) and, along with urine samples, will be analysed for F2-isoprostanes as a marker of oxidative stress, as well as NO2- and NO3- levels.54 55 (link) Serum samples will be analysed for LDH and CK, as markers of muscle damage using an automated analyser with commercial kits (Konelab 20XTi, Thermo Fisher Scientific, Waltham, Massachusetts, USA).
5
Blood Sampling for Metabolic Measures
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An intravenous catheter (BD Nexiva catheters; 20GA 1.25IN 1.1 × 32 mm, Becton Dickinson and Co., Franklin Lakes, NJ, USA) was placed in a large antecubital vein. Blood samples were taken at −5, 0, 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, and 180 min into both a tube with a sodium fluoride EDTA (ethylenediaminetetracetic acid) and a tube with no additives. The tube with no additives for serum insulin, C-peptide and triglyceride (TG) was stood upright at room temperature for 30 min, and then it was placed on ice, whereas the sodium fluoride EDTA tube for plasma glucose was placed straight on ice until centrifugation and processing. Blood samples were centrifuged at 4000 rpm at 4 °C for 10 min (Universal 32R, Hettich Zentrifugen, Tuttlingen, Germany). Plasma and serum aliquots were stored at −80 °C until analysis. The measurements of plasma glucose and serum TG were made using an automated spectrophotometric analyzer (Konelab 20XTi, ThermoFisher Scientific, Waltham, MA, USA). Serum insulin and C-peptide analyses were conducted by commercial ELISA kits (Alpha Diagnostic, San Antonio, TX, USA, Kit # 0030N for insulin, Kit # 0040 for C-peptide).
6
Comprehensive Metabolic Profiling Protocol
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Fasting venous blood will be drawn by venepuncture by a trained phlebotomist to measure lipid profiles, glucose, inflammatory markers, erythrocyte fatty acids and genetic risk profiles. A total of 60 mL will be collected, centrifuged (4°C, 4000 rpm, 10 min) and frozen at −80°C for batch processing at the end of each data collection time point. Blood samples will be analysed in duplicate with commercial assay kits (including quality controls and calibrators) using the KONELAB 20XTi (ThermoFisher, Massachusetts, USA).
7
Quantification of Phosphoglucomutase Activity
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Phosphoglucomutase activity was measured in cell lysates from control hiPSCs (hiPS-1, hiPS-2) and from patient-derived PGM1-deficient hiPSCs (hiPS_PGM1-1, hiPS_PGM1-2) using a Konelab 20XTi (Thermo Fisher Scientific, Waltham, MA, USA). Cells were washed with 1X PBS (Gibco, Life Technologies, Carlsbad, CA, USA), detached with 0.5 mM EDTA solution (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), and pelleted via centrifugation at 550× g for 5 min at room temperature. Next, the pellets were washed with 6 mL of 0.9% NaCl three times, and afterwards the dry pellets were snap-frozen in liquid nitrogen and stored at −80 °C till measurement. The enzyme activity assay performed was based on the quantification of NADPH production, measured by absorbance at 340 nm according to Van Schaftingen and Jaeken [54 (link)]. Data on PGM activity were normalized on protein content, also measured using a Konelab 20XTi. Another enzyme, namely phosphomannose isomerase (PMI), was used as control. Data analysis and visualization were performed in Microsoft Office Excel and PRISM GraphPad 5.03.
8
Analytical Methods for Food Composition
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The standard parameters were established as follows: total soluble solids (TSS) was measured by a digital automatic refractometer (Abbemat, Anton Paar®, St. Albans, Austria). The total saccharide content was enzymatically determined using a Konelab 20XTi analyzer (Thermo Fisher Scientific, Schwerte, Germany) and its proper kits (EnzytecTM fluid, Thermo Fisher Scientific). Total acidity and pH were analyzed by a Schott titrator (Titroline alpha plus, SI-Analytics, Texas City, TX, USA). The content of total phenols was evaluated by the Folin assay (Singleton and Rossi 1965) using an automatic analyzer (Konelab 20XTi, Thermo Fisher Scientific). Amino acids were determined by an Amino Acid Analyzer S433 (Sykam GmbH, Eresing, Germany). All measurements were made in triplicate.
9
Comprehensive Cardiometabolic Risk Assessment
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Body height will be measured with the head positioned in the Frankfurt plane with a wall-mounted stadiometer and body composition with a bioelectrical impedance device (InBody 720, Seoul, South Korea; [113 (link)]). Venous blood samples will be drawn after standard overnight (12 h) fast. Serum insulin will be measured by electrochemiluminescence immunoassay (Immulite 2000, Siemens, Germany). Fasting plasma glucose, total plasma cholesterol, plasma high and low-density cholesterol and plasma triglycerides will be measured by enzymatic colorimetric assays (Konelab 20XTi, Thermo, USA). Insulin sensitivity estimated by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) will be calculated using the formula: fasting serum insulin x fasting plasma glucose/22.5 [114 (link)]. White and red blood cells, hematocrit and hemoglobin will be measured by an automated analyzer (Sysmex XP300, Sysmex, Japan). Systolic and diastolic blood pressure, aortic pulse wave velocity, and augmentation index will be measured in supine position with a non-invasive oscillometric device after 10 min of rest (Arteriograph, Tensiomed Ltd., Budapest, Hungary; [115 (link)]). Participants will be allowed to drink water and take their usual medications during fastening.
10
Multiparametric analysis of mussel biomarkers
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Hemocyte viability, phagocytosis, ROS production, lysosomal system size and CSP-3 induction were measured by flow-cytometry (FACScaliburTM, BD Biosciences) using protocols adapted from Minguez et al. (2012) (link).
Prior to biomarker analysis on the automated chemistry analyzer Konelab 20 Xti (Thermo Scientific), digestive glands were treated as described in Sroda and Cossu-Leguille (2011) (link). Antioxidant and antitoxic defenses, i.e. total antioxidant capacity (TAC; Erel (2004) (link)), total glutathione peroxidase (GPx), glutathione-S-transferase (GST) and the acid phosphatase activities (ACP) were measured using colorimetric methods adapted and developed on the Konelab. Catalase activity (CAT) was measured spectrophotometrically (Beer and Sizer, 1952) . Lipid hydroperoxide concentration ([LOOH]) was measured following the automated method developed by Arab and Steghens (2004) (link) and Caspase-3 activity (CSP-3) activity was assessed following manufacturer instructions (Euromedex). Protein, triglyceride, cholesterol concentrations ([prot], [trigly.] and [chol.]) and Lactate Dehydrogenase (LDH) activity were measured using Thermo-Scientifc Konelab ready-to-use reagents. Electron Transport System (ETS) mitochondrial activity was measured following the method from Coen and Janssen (1997) (link). Finally, mussel filtration rate was measured according to Palais (2011) .
Prior to biomarker analysis on the automated chemistry analyzer Konelab 20 Xti (Thermo Scientific), digestive glands were treated as described in Sroda and Cossu-Leguille (2011) (link). Antioxidant and antitoxic defenses, i.e. total antioxidant capacity (TAC; Erel (2004) (link)), total glutathione peroxidase (GPx), glutathione-S-transferase (GST) and the acid phosphatase activities (ACP) were measured using colorimetric methods adapted and developed on the Konelab. Catalase activity (CAT) was measured spectrophotometrically (Beer and Sizer, 1952) . Lipid hydroperoxide concentration ([LOOH]) was measured following the automated method developed by Arab and Steghens (2004) (link) and Caspase-3 activity (CSP-3) activity was assessed following manufacturer instructions (Euromedex). Protein, triglyceride, cholesterol concentrations ([prot], [trigly.] and [chol.]) and Lactate Dehydrogenase (LDH) activity were measured using Thermo-Scientifc Konelab ready-to-use reagents. Electron Transport System (ETS) mitochondrial activity was measured following the method from Coen and Janssen (1997) (link). Finally, mussel filtration rate was measured according to Palais (2011) .
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