Ultrapure concentrated nitric acid

Sample collection and processing were carried out in local clinics. Five milliliters of venous blood was collected in a pro-coagulation tube, let stand at room temperature for 15 min, and centrifuged at 12,000 rpm for 10 min to separate serum, and immediately transferred to 2 mL frozen pipe. A spot 10-mL urine sample collected from each subject at the end of a work shift have been described previously [10 (link)]. All samples were stored at -80°C until analysis. All test tubes used in the study were free of metal contamination.
The 28 elements analyzed in this work were aluminum (Al), arsenic (As), barium (Ba), beryllium (Be), bismuth (Bi), cadmium (Cd), calcium (Ca), cesium (Cs), chromium (Cr), cobalt (Co), copper (Cu), indium (In), iron (Fe), lead (Pb), lithium (Li), manganese (Mn), magnesium (Mg), molybdenum (Mo), nickel (Ni), potassium (K), rubidium (Rb), selenium (Se), silver (Ag), sodium (Na), strontium (Sr), thallium (Tl), vanadium (V), and zinc (Zn).
At the time of sample analysis, serum and urine samples were brought to room temperature. An aliquot of 500 μL serum and urine samples was diluted with a solution containing 0.1% (V/V) Triton-X-100 (Sigma, USA) and 1% ultrapure concentrated nitric acid (Sigma, USA) to a 5 mL total volume. Prior dilution of each sample was critical in order to obtain the best results. The samples were then quantified by ICP-MS using freshly made multi-element stock solution on the day of analysis. The instrument parameters are as followed: nebulizer carrier gas flow, 1.10 L/min; sample depth, 4.8 mm; RF power, 1450 W; sampler/skimmer, nickel; CeO⁺/Ce⁺, <0.5%; ¹⁴⁰Ce¹⁶O/¹⁴⁰Ce, <2%. Calibration was performed using a certified reference standard (Agilent, USA). Validity of the calibration curve was evaluated by analyzing the standards from the same source after every two hours injection. The calibration curve was considered valid if the observed concentration of the independent standard was within 10% of the expected concentration. We also measured the recovery of each sample at random. The internal standard and the limits of detection (LOD) for these 28 elements were shown in Table 1.

The reagents purchased from the Merck Company (Darmstadt, Germany) were: ultrapure concentrated nitric acid (69%), standard solutions of studied elements—arsenic (H₃AsO₄ in HNO₃ 0.5 mol L⁻¹, 1000 mg L⁻¹ As), cadmium (Cd(NO₃)₂ in HNO₃ 0.5 mol L⁻¹, 1000 mg L⁻¹ Cd), lead (Pb(NO₃)₂ in HNO₃ 0.5 mol L⁻¹, 1000 mg L⁻¹ Pb), mercury (Hg(NO₃)₂ in HNO₃ 2 mol L⁻¹, 1000 mg L⁻¹ Hg). From Sigma-Aldrich (Saint Louis, MO, USA) ammonium dihydrogen phosphate was obtained. Certified reference material mixed polish herbs (INCT-MPH-2) was obtained from the Institute of Nuclear Chemistry and Technology (Warsaw, Poland).

Trained clinical staff collected whole blood samples from participants during their study visit using standard protocols. Trace metal-free vacutainers (Becton-Dickinson, Franklin Lakes, NJ, USA) were used to limit external metal contamination. Samples were frozen at −20 °C and stored until analysis. Whole blood samples were analyzed for Mn at the Chinese Centers for Disease Control and Prevention in Beijing using inductively-coupled mass spectrometry (ICP-MS); these methods have been described previously [45 (link),46 (link)]. Briefly, blood samples were diluted with 0.01% Triton-X-00 (Sigma-Aldrich, Saint Louis, MO, USA) and 0.5% ultrapure concentrated nitric acid (Merck, Darmstadt, Germany). Samples were vortexed and then analyzed using XSERIES 2 ICP-MS (Thermo Fisher, Waltham, MA, USA). The SeronormTM Trace Elements Whole Blood Control Level 1 (Sero AS, Billingstad, Norway) was used for internal quality insurance. None of the collected samples had a Mn concentration below the detection limit (DL) of 0.11 μg/L.
Although toenail Mn is more commonly used than fingernail Mn, FMn has been used in previous studies as a biomarker of exposure [47 (link)]. Fingernails were used in this study due to a concern that toenails would have a greater potential for external contamination because participants were observed to wear open-toed shoes to the factory. Participants were asked to thoroughly wash their hands with soap to remove external debris. Fingernail samples from participants’ 10 fingers were collected using a titanium dioxide nail clipper. Fingernail samples were stored in small Ziploc bags and kept at room temperature until analysis. Samples were cleaned twice using ultrasonic cleaning procedures in 1% Triton X-100 solution (Sigma-Aldrich Inc., Saint Louis, MO, USA) [48 (link)]. Following each cleaning, the nails were rinsed multiple times with deionized (DI) water and then dried at 60 °C. After the second round of cleaning, the fingernail samples were digested at 200 °C in ultrapure nitric acid (Sigma-Aldrich Inc., Saint Louis, MO, USA), then analyzed for Mn using the ELEMENT-2 mass spectrometer (ThermoFinnigan, Bremen, Germany) at Purdue University’s Campus-Wide Mass Spectrometry Center. Mn concentrations were corrected for systematic error using an internal standard run simultaneously with the samples. The DL for fingernail Mn ranged from 1.31 to 3.97 ppb. Seventeen (28.3%) FMn measurements were below the DL but still had detectable concentrations which were larger than blank samples. Replacement of values

link),50 (link)]. While these values likely have a greater variability and uncertainty compared to values >DL, this approach is likely to reduce the potential for left-censoring to induce bias in our results.
After fingernails were cut but prior to the BnMn measurement, participants were asked to wash their right hand and lower arm with soap and water for a second time. A trained research assistant then cleaned participants’ right hand and lower arm with 50% alcohol wipes. Each participant’s right hand was irradiated for 10 min to excite the ⁵⁵Mn atoms in the hand bone to ⁵⁶Mn. A bag filled with water was wrapped around the participant’s arm to hold it in place as well as reduce the whole body effective radiation dose (estimated at 23 µSv) [32 (link)]. After 5 min of rest to allow for the initial decay of unstable isotopes, participants then moved to a high purity germanium (HPGe) detection system that collected Mn γ ray (847 keV) spectra over the course of an hour as the ⁵⁶Mn neutrons de-excite [32 (link)]. BnMn concentrations were calculated from the Mn γ ray spectra using a pre-existing calibration line created from a set of Mn-doped bone-equivalent hand phantoms. A Mn/Ca γ ray ratio was used to account for variation in neutron flux, hand palm attenuation, and counting geometry. This also accounts for bone density, making this a comparable measure to BnMn presented in µg/g Ca. BnMn in µg/g multiplied by 3.94 is equivalent to BnMn in µg/g Ca [33 (link),34 (link)]. The transportable IVNAA system has a DL of 0.64 µg Mn per g dry bone (ppm) [33 (link)]. There were 19 (31.7%) participants with BnMn < DL; 13 of the 19 (68.4%) were negative values. Negative BnMn measurements can be estimated when the true BnMn value is close to zero: this has also been seen in bone lead measurements [51 (link)]. Similar to the rationale described above for retaining fingernail Mn measurements

link),52 (link)]. Therefore, these values were included in the current analysis.