Ultrapure hno3

One mL of whole blood from each subject was collected into a 15 mL polystyrene tube (Corning, Glendale, AZ, USA), and was added to 2 mL of ultrapure HNO₃ (VWR, Leuven, Belgium) and digested on a heat block (ModBlock, CPI International, Santa Rosa, CA, USA) at 80 °C until complete dissolution. The digests were further diluted with ultrapure deionized water (Micro Pure UV, Thermo Scientific Barnstead, Langenselbold, Germany).
Approximately 0.150 g of cancer and non-cancer tissues were dried at 105 °C overnight (the water content was ca. 50%). Further, samples were weighed in 15 mL polystyrene tubes, added with 2 mL of ultrapure HNO₃ (VWR), digested on a heat block at 80 °C until complete dissolution (ModBlock), and then diluted with ultrapure, deionized water for analysis.

For these experiments, mycelia of the different strains were produced in Potato Dextrose Broth (PDB, Difco ® MD, USA) by growing them at 30 °C for up to 8 days (depending on the growth speed of the strain). Thirty grams of mycelium was inoculated in PDB in the presence (or absence as a control) of heavy metals (100 ppm of Cu; 100 ppm of Zn; or 350 ppm of Pb, each in separate tubes with fresh PDB or in a tube containing a mixture of 100 ppm of each metal). A two mL aliquot was taken from these cultures every 6 hours, centrifuged, and the supernatant was subjected to atomic absorption spectrophotometry for metal content, using an atomic absorption spectrophotometer (908AA, GBC Scientific Equipment Pty Ltd., GBC, Dandenong, Australia), with background correction. All the material was previously washed with ultrapure HNO₃ (J.T. Baker ® NJ, USA) for 24 hrs. To ensure a satisfactory accuracy of the analysis, standard reference material of the National Institute of Technology and internal reference materials were used for precision, quality assurance, and control for selected metal measurements. For each measurement, average values of three replicates were recorded.
Metal content is reported as ppm (parts per million). Detection limits of the atomic absorption spectrophotometer are Zn: 0.0005mg/L, Pb: 0.01 mg/L, and Cu: 0.001 mg/L.
pH was measured at the end of the experiment.

HCl and HNO₃ for sample preparation and dilutions were BAKER INSTRA-ANALYZED Reagent (JT Baker, USA), and H₂O₂ was an optima grade from Fisher Chemical (UK). Ultrapure HNO₃ (Ultrex II grade, JT Baker, USA) was used for Tl solution preparation. Tin (II) chloride dihydrate (SnCl₂·2H₂O) was of reagent grade from Scharlau (Spain). All aqueous solutions were prepared using ultrapure water (18 MΩ, Millipore, USA).