Cobas 8000 analyzer system

0.1 mM CuCl₂ and oleic acid (125 μM; O3008, Sigma-Aldrich) were added in standard cell culture medium for 24 h. Cells were subjected to TG quantification (Triglyceride Quantification Assay, ab65336, Abcam) or AdipoRed Assay Reagent (PT-7009, Lonza) according to the protocol of the manufacturer. Total protein concentration was used for normalization. Cell culture supernatant was collected and triglycerides were determined by an automated cobas 8000 analyzer system (Roche Diagnostics GmbH, Mannheim, Germany).

Parameters for complete blood count (CBC) were assessed using hematology analyzer (Hospitex Diagnostic, Italy) (Bain et al., 2016 ). Biochemical markers such as total protein, albumin and glucose levels were assessed using kits from Bio-diagnostic, Giza, Egypt (Wang et al., 2010 ). Globulin levels were detected by subtracting albumin from the total protein, and A/G values were calculated by dividing albumin to globulin. According to the provided instructions, total bilirubin (MBS730053) and direct bilirubin (MBS1607953) were assessed using ELISA kits (MyBioSource, San Diego, USA). Liver enzymes including aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALKP), gamma glutamyl transferase (γGT) and lactate dehydrogenase (LDH) were measured using kits (Bio-diagnostic) (Murray and Kaplan, 1984 , Burtis and Ashwood, 1994 ). In addition, total cholesterol (TC), triglycerides (TG), in addition to high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (HDL-C) measurements were taken using Roche Cobas 8000 analyzer system (Mannheim, Germany).

In the LOOP Study, whole-blood samples were taken from study participants at the local centers upon randomization. NT-proBNP levels were measured in 2 central hospital laboratories with the sandwich electrochemiluminescence immunoassay (Roche Diagnostics GmbH, Germany) and the Cobas 8000 analyzer system (Roche Diagnostics) in accordance with manufacturer instructions.
In the present study, we included the participants with available NT-proBNP measurement at baseline and further divided them into 2 subgroups based on the median NT-proBNP level, which was also identical to the clinically reasonable cutoff for AF screening, as previously proposed and currently used in ongoing trials.^{13 (link),14 (link)} Supplementary analyses were further conducted to assess NT-proBNP as a continuous variable. NT-proBNP levels are reported as picomole per liter (pmol/L). The conversion factor for NT-proBNP expressed as picogram per milliliter (pg/mL) is 1 pg/mL=0.12 pmol/L (Table S1).^{15 (link)}

At week 9, serum carbamide and creatinine levels were measured to assess renal function. Serum carbamide and creatinine levels were quantified by kinetic UV method using urease and glutamate dehydrogenase enzymes and Jaffe method, respectively. All reagents and instruments for the serum parameter measurements were from Roche Diagnostics (Hoffmann-La Roche Ltd., Basel, Switzerland), as described previously [76 (link)]. Cardiovascular risk factors, including serum cholesterol and triglyceride levels, were also measured by Roche Cobas 8000 analyzer system using enzymatic colorimetric assays from Roche (Hoffmann-La Roche Ltd., Basel, Switzerland) [29 (link)].

At weeks 5 and 12, animals were placed into metabolic cages (Tecniplast Metabolic Cage System, Buguggiate, Italy) for 24 h to measure urine creatinine and protein concentrations by standard laboratory methods as described previously^{35 (link)–37 (link),52 (link)} (Fig. 1). Blood was collected from the saphenous vein at week 4 and from the abdominal aorta at week 13 to measure serum carbamide (urea), creatinine, and lipid levels to verify the development of CKD^{35 (link)–37 (link),52 (link)}. Serum urea and creatinine levels were quantified by kinetic UV spectrophotometric method using urease and glutamate dehydrogenase enzymes and Jaffe’s method, respectively, using the Roche Diagnostics reagents and platform analyzers (Hoffmann-La Roche Ltd., Basel, Switzerland)^{35 (link),52 (link)}. At week 5 and the endpoint, urine creatinine and volume and serum creatinine concentration were measured^{35 (link),52 (link)}. Creatinine clearance, a renal functional indicator, was calculated using the standard formula (urine creatinine concentration [μM] × urine volume for 24 h [mL])/(serum creatinine concentration [μM] × 24 × 60 min)^{40 (link),52 (link)}. At weeks 5 and 13, total serum cholesterol and triglyceride levels were measured by Roche Cobas 8000 analyzer system using enzymatic colorimetric assays (Roche, Hoffmann-La Roche Ltd., Basel, Switzerland)^{36 (link)}.

Serum carbamide and creatinine levels were quantified by kinetic UV method using urease and glutamate dehydrogenase enzymes and Jaffe method, respectively. Serum sodium, potassium, and chloride levels were determined by indirect potentiometry using ion-selective electrodes. The serum calcium, magnesium, phosphate levels were quantified by complex formation methods. All reagents and instruments for the serum parameter measurements were from Roche Diagnostics (Hoffmann-La Roche Ltd, Switzerland), as described previously^{33 (link),35 (link)}. Urine creatinine and urine protein levels were measured by standard laboratory methods as described previously^{33 (link),35 (link)}. At week 13, serum total cholesterol, HDL-cholesterol and triglyceride levels were measured by Roche Cobas 8000 analyzer system using enzymatic colorimetric assays from Roche (Hoffmann-La Roche Ltd, Switzerland)^{65 (link),66 (link)}. LDL-cholesterol was calculated according to the Friedewald formula. Total blood count and hematocrit were measured from whole blood by a hematology analyzer (XE-2100, Sysmex Corporation, Japan) at week 13 to verify the development of chronic systemic inflammation and renal anemia.