Urine was collected and tissue extracts were prepared by sonication in phosphate buffer saline from the wild type (n = 4) and IDUA KO (n = 4). The tissue extracts were prepared by sonication in phosphate buffer saline. Extracts were centrifuged at 20,000 g for 30 min, and supernatants were collected. GAGs concentrations in urine and tissue were quantified via colorimetric assay (Kamiya Biomedical Co., USA) according to the manufacturer’s instructions, and absorbance was measured at 620 nm using a chondroitin 6-sulfate standard curve. Tissue GAG contents were normalized to the total protein concentration measured using a BCA assay (Pierce, Rockford, IL, USA), and urinary GAG excretion was normalized to creatinine by creatinine assay (Sigma-Aldrich, St Louis, MO, USA).
Creatinine assay
Manufactured by Merck Group
Sourced in United States, Germany
The Creatinine assay is a laboratory test used to measure the concentration of creatinine in a biological sample, typically blood or urine. Creatinine is a byproduct of muscle metabolism and its measurement is important for assessing kidney function.
Automatically generated - may contain errors
4 protocols using creatinine assay
1
Glycosaminoglycan Quantification in Urine and Tissues
2
Creatinine and Albumin Quantification
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Serum/urinary creatinine and urinary albumin were measured using creatinine assay (Sigma–Aldrich) kits and mouse albumin ELISA kits (Bethyl Laboratories) according to the manufacturers' protocols.
3
Biomarker Collection and Storage
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Blood was collected periodically throughout the study via submandibular puncture using sterile 5 mm lancets (Goldenrod) into microvette heparinized tubes and centrifuged in an Eppendorf centrifuge 5415D at 7000 rpm for 10 min. Plasma was collected and stored at −20 °C to −80 °C for IDS assay. Urine was collected periodically and stored at −20 °C until assay for GAG (Accurate Chemical, NY) and creatinine assay (Sigma-Aldrich, MO).
4
Phthalate Exposure and Metabolic Biomarkers
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Mono(2-ethylhexyl) phthalate (CAS No. 4376-20-9, 97 % purity) and monobutyl phthalate (CAS No. 131-70-4, 97 % purity) as well as the following kits – alanine aminotransferase (ALT) activity assay (catalogue No. MAK052), aspartate aminotransferase (AST) activity assay (catalogue No. MAK055), urea assay (catalogue No. MAK266), triglyceride quantification (catalogue No. MAK006), bromocresol purple albumin assay (catalogue No. MAK125), creatinine assay (catalogue No. MAK080), total protein kit (catalogue No. TP0100), and glucose assay kit (catalog No. CBA086) – were obtained from Sigma-Aldrich (Steinheim, Germany). To measure insulin levels, we used the insulin rat ELISA Kit (Thermo-Fisher Scientific, Waltham, USA). Rat islet cell antibody (catalogue No. BG-RAT11369) was obtained from Novatein Biosciences (Woburn, MA, USA), and rat insulin antibody (catalogue No. RAB0904) from Sigma-Aldrich.
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