Optima grade

Urinary metals/metalloids were detected with the Octagon-based Collision/Reaction Cell (Agilent Technologies) and Agilent 7700X Inductively Coupled Plasma Mass Spectrometer (ICP‒MS).
Morning urine samples of subjects were collected in a microelement-free container and stored at − 20 °C. Samples were pretreated according to the following steps. Then, 500 μL urine was added to 20 μL of 67% (Vol/Vol) HNO₃ (OptimaTM grade; Fisher Scientific) before being placed into a refrigerator overnight for digestion. Then, 0.5 ml of each sample was mixed and diluted tenfold with 1% (Vol/Vol) HNO₃. A total of 21 metal concentrations were finally determined: aluminum (Al), zinc (Zn), argentum (Ag), As, barium (Ba), Cd, copper (Cu), iron (Fe), Hg, cobalt (Co), chromium (Cr), cesium (Cs), Mn, Se, strontium (Sr), nickel (Ni), Pb, rubidium (Rb), thallium (Tl), uranium (U), and vanadium (V). Instrument performance was verified each time by applying standard reference materials (SRMs) 1640A (National Institute of Standards and Technology, Gaithersburg, MD, USA) and a blank control (1% HNO₃). The spiked recoveries of quality control standards ranged from 85 to 120%, and the limits of detection (LODs) varied between 0.00004 μg/L and 0.25 μg/L. Moreover, we used a fully automatic biochemical analyzer (Mindray Medical International Ltd.) to detect the urinary specific gravity (SG) of each sample.

The determination of metal contents in urine was performed as previously described [37 (link)], with minor modification. In brief, the frozen urine samples were completely thawed at room temperature and homogenized. A 3.0 ml aliquot of urine was transferred to a polypropylene tube (Jiayu experiment instrument Co., Ltd., Haimen, China) containing 15.0 μl of 67% (v/v) HNO₃ and stored in a refrigerator at 5°C. Two hours before sample preparation the urine samples were brought to room temperature. A 1.0 ml of the sample was pipetted into a 10ml disposable polypropylene tube and then filled up to 5.0 ml with 1.2% (v/v) HNO₃ (OptimaTM grade, Fisher, Belgium) using adjustable volume pipette samplers. The samples were then measured using an inductively coupled plasma mass spectrometry with an octopole based collision/reaction cell (Agilent 7700 Series, Waldbronn, USA).

Fisher Optima^TM grade (Fisher Scientific, Hanover Park, IL, USA) solvents were used for sample preparation. Internal standards were obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA) (citric acid-d₄, dl-glutamic acid-d₅, d-sorbitol-¹³C₁, and l-leucine-d₃) or Sigma-Aldrich (St. Louis, MO, USA) (4-nitrobenzoic acid). Standards for obtaining retention indices (C₄–C₂₄ FAMEs, methyl nonanoate, and alkane standard mix (C₁₀–C₄₀)) and for urine pretreatment (urease from Canavalia ensiformis (Jack bean) type III) were obtained from Sigma-Aldrich. Derivatization chemicals were obtained from Thermo Scientific (Waltham, MA, USA) (Methoxamine (MOX) reagent and N-methyl-N-[trimethylsilyl]trifluoroacetamide (MSTFA) with 1% (v/v) trimethylchlorosilane (TMCS)).

The deionized water was prepared using a Direct Q 3UV purification system (Merck). Methanol and acetonitrile (Optima grade) were obtained from Thermo Fisher Scientific, ammonium carbonate; 25% ammonia solution, 4-fluorophenylalanine, 1C6-glucose-6-phosphate, and 2-dipalmitoyl-sn-glycero-3-O-4′-[N,N,N-trimethyl(d9)]-homoserine (d9-DGTS) from Merck; and hexakis(2,2-difluoroethoxy)phosphazene and tris(trifluoromethyl)-1,3,5-triazene from Apollo Scientific.

Hepatic and skeletal muscle iron pool has been quantified on the ‘Analyse Elémentaire et Métabolisme des Métaux’ (AEM2) Platform (Université de Rennes 1—Centre Hospitalier Universitaire). Frozen skeletal muscle and liver samples were desiccated at 120°C for 15 h in an overnight. They were weighed and mineralized according to a previous protocol.²⁶ Briefly, nitric acid solution (Optima Grade, Thermo Fisher Scientific, Waltham, MA, USA) was added to dried samples, and then the tubes were placed in a MARS6 microwave (CEM, Matthews, NC, USA) with a temperature maintained at 180°C. Iron (⁵⁶Fe) was quantified by inductively coupled plasma mass spectrometry on an X‐Series IO from Thermo Fisher Scientific equipped with collision cell technology (ÆM2 platform). Calibration ranges were prepared using a multi‐element calibrator solution (Plasma Cal, SCP SCIENCE, Baie‐D'Urfe, QC, Canada).

Freshly passaged C. parvum oocysts were purchased from Bunch Grass Farm (Deary, ID) and handled under BSL-2 protocols, with the approval of the Boston University Institutional Biosafety Committee. All reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless noted otherwise. Solvents used for LC-MS were Optima™ grade, procured from Fisher Scientific (Thermo-Fisher Scientific, Waltham, MA).