Creatinine anhydrous

Lead nitrate (Pb(NO₃)₂), sodium lauryl sulfate, creatinine anhydrous, propionic acid, lithium d-, and l-lactate, acetylacetone, 37% formaldehyde, cimetidine, indoleacetic acid, methylumbelliferone, and methylumbelliferyl N-acetyl-β-d-glucosaminide were purchased from Sigma-Aldrich Fine Chemicals Inc. (MO, USA). Formic acid, HPLC grade acetonitrile (ACN), ethanol and methanol (MeOH) were purchased from Merck (Darmstadt, Germany). Hippuric acid, 2,2′-dipyridyl disulfate (DPDS), triphenylphosphine (TPP) and 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 5-Aminolevulinic acid hydrochloride and guanidinoacetic acid were purchased from Alfa Aesar (MA, USA). Sulbactam was purchased from Cayman Chemical (Michigan, USA). Acetic acid was purchased from Hayashi Pure Chemical Ind.,Ltd. (Osaka, Japan). Trifluoroacetic acid (TFA) was purchased from Riedel-de Haën (Seelze, Germany). 3-(Trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt (TSP) was purchased from Cambridge Isotope Laboratories (MA, USA). 1,1-Dimethylbiguanide hydrochloride (metformin) was purchased from Santa Cruz Biotechnology, Inc. (Texas, USA). Protein Assay Dye Reagent was purchased from Bio-Rad Laboratories (California, USA).

Urinary albumin excretion was determined by Mouse Albumin ELISA quantification Kit (Bethyl laboratories, Inc., Montgomery, TX, USA) according to the manufactory's instruction.
Urine creatinine was measured by isocratic high-performance liquid chromatography (HPLC) on a Zorbax SCX300 column (Agilent, USA) using a slight modification of a method first reported by Yuen et al. [33 (link)]. In brief, 5 μL urine was added to 100 μL acetonitrile containing 0.5% acetic acid and vortexed for 15 seconds to extract the creatinine. After 15 min of −20°C storage and centrifugation the supernatants were evaporated and then reconstituted with 25 μL 5 mM sodium acetate, pH 4.1. The samples were centrifuged for 10 min at 3000 rpm. Duplicate samples (10 ul each) were fractionated on a 50 mm × 2.1 mm Zorbax SCX300 column with an in-front SCX guard column. Isocratic HPLC was performed at a flow rate of 1 mL/min, and UV absorbance was monitored at 225 nm. A standard curve was created by including a 2-fold dilution series of creatinine anhydrous (Sigma Aldrich). All aqueous solutions were filtered through a 0.22-micron filter before use.

Standards of sarcosine 98 %, α-alanine ≥ 99 % and β-alanine (BioUltra) ≥ 99.0 % (NT), glycine ≥ 99 % (HPLC), creatinine anhydrous > 98 % purity and ammonium chloride 99.5 %, A.C.S. were purchased from Sigma-Aldrich Corporation (Sigma-Aldrich, Saint Louis, MO, USA). N, O-Bis-(trimethylsilyl) trifluoroacetamide with 1 % trimethylchlorosilane [BATFA + TMCS, 99:1 (Sylon BFT)], derivatization grade for GC, was obtained from SUPELCO (Bellefonte, USA). Acetonitrile and water for chromatography grade were procured from Merck (Darmstadt, Germany). Urea 99.5 % AR and potassium phosphate dibasic AR were purchased from Loba Chemie Pvt. Ltd., (Mombai, India). Potassium chloride, calcium chloride dehydrate, sodium sulfate anhydrous, and sodium chloride were purchased from Merck.