Labassay creatinine

Blood ammonia levels were determined using the bromocresol green method (PocketChem^TM, PA-4140, Arkray, Inc., Kyoto, Japan, measurement range: 10 to 400 nitrogen-µg/dL) or, if the values exceed the detection range of the method, the indophenol method (Ammonia-Test-Wako kit [277-14401], FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The values by the bromocresol green method were compensated by multiplying by 1.25, which is the ratio of molecular weights of nitrogen and ammonia. Arterial oxygen saturation was measured using a portable blood analyzer, iStat, and its cartridge, CG4+ (Abbott, Abbott Park, IL, USA). Urinary orotic acid levels and hepatic OTC activities were determined using a colorimetric assay [6 (link)]. Urinary orotic acid levels were standardized with urinary creatinine levels measured using a laboratory assay kit (LabAssay™ Creatinine [290-65901], FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). OTC activities were assayed with homogenates prepared from the liver of piglets of OTCD (n = 6) and wild-type male piglets (n = 5) just after birth (not autopsied liver), and they were expressed as ng citrulline production/min/mg protein. Protein content was determined using the Bradford method (Bio-Rad Protein Assay [5000001JA], Bio-Rad Laboratories, Inc. Hercules, CA, USA).

Plasma creatinine was measured by a commercially available kit (LabAssay Creatinine, Wako Pure Chemical Industries Ltd., Osaka, Japan). For hematocrit measurement, blood was collected in hematocrit tubes followed microhematocrit centrifugation. The length of the column of packed red cells was measured, divided by the length of the column of whole blood and multiplied by 100%. Levels of plasma BUN and hemoglobin (Hb), as well as total protein in urine were measured by an automated analyzer (7020-Automatic Analyzer; Hitachi High-Technologies, Tokyo, Japan). Plasma erythropoietin (EPO) level was measured in both mice and rats by ELISA (Epo Mouse ELISA Kit, Cat. No. KA1998, Abnova, Tapei City, Taiwan; Legend Max Rat Erythropoietin ELISA Kit, Cat. No. 442807, BioLegend, San Diego, CA, USA, respectively) in accordance with the manufacturers’ instruction. Levels of ferritin and gamma glutamyltransferase (γ-GT) were also measured in rat plasma by commercially available ELISA kits (Ferritin (Rat) ELISA kit, Cat. No. KA1949, Abnova; Rat γ-GT1 ELISA Kit, Cat. No. E-EL-RO404, Elabscience, Wuhan, China, respectively).

Body weights were recorded, and blood pressures and pulse rates were measured using the tail-cuff method with an automatic sphygmomanometer (BP98A; Softron Co., Ltd., Tokyo, Japan). The 24 h urine samples were collected on the day before euthanasia. Urinary albumin levels were determined by ELISA using a murine microalbuminuria ELISA kit (Albuwell M; Exocell, Inc., Philadelphia, PA, USA). Urinary creatinine was measured using commercially available LabAssay Creatinine (Wako Pure Chemical Industries, Osaka, Japan). Serum creatinine levels were measured at a central clinical laboratory (SRL, Inc., Tokyo, Japan).