Suprapur

Kjeldahl tablets were purchased from Merck (Darmstadt, Germany). The dietary fiber assay kit and celite were provided by Sigma-Aldrich (St. Louis, MO, USA).
For mineral analysis, nitric acid (≥69% (w/w) Trace Metal™ was purchased from Fisher Scientific (Leicestershire, UK), hydrogen peroxide (30% w/w) Suprapur^® was provided by Merck (Darmstadt, Germany), and hydrochloric acid (>30% w/w) TraceSELECT™ was supplied by Honeywell Fluka™ (Seelze, Germany). Certified reference materials (BCR-679, ERM-BB422, ERM-BC382) were provided by the European Commission, Joint Research Centre (Brussels, Belgium). A 10 µg/mL internal standard (Mix1-SCP-IS7) and single-element stock solutions (1000 µg/mL) of gallium (Ga), rhodium (Rh), and calcium (Ca) were from SCP SCIENCE (Quebec, QC, Canada); single-element stock solutions (1000 µg/mL) of iron (Fe), magnesium (mg), sodium (Na), and potassium (K) for Atomic Absorption Spectroscopy were from Fluka (Seelze, Germany); Sigma-Aldrich (Buchs, Switzerland) provided phosphorus (P) and mercury (Hg) single-element standards (1000 µg/mL); and the ICP multi-element standard solution (21 elements) was obtained from Supelco (Darmstadt, Germany).
For chromatographic analyses, the following standards/reagents were used: standards of amino acids (98.0%–≥99.5%), tocopherols (α-, β, λ-, and δ-), fructose, glucose, and sucrose, as well as the Supelco 37 Component FAME Mix, were all provided by Sigma-Aldrich (St. Louis, MO, USA); Larodan (Solna, Sweden) provided tocotrienols (α-, β-, λ-, and δ-) and tocol; L-norvaline was purchased from Sigma (Deisenhofen, Germany); caffeine and chlorogenic acids (3-, 4-, and 5-caffeoylquinic acids) were supplied by Honeywell Riedel-de Haën (Seetze, Germany). The derivatization reagents (9-fluorenylmethyl chloroformate and o-phthalaldehyde/3-mercaptopropionic acid) were purchased from Agilent Technologies (Palo Alto, CA, USA). For eluent preparation, 1,4-dioxane was obtained from Sigma-Aldrich (St. Louis, MO, USA); HPLC-grade methanol, acetonitrile, and n-hexane were acquired from Honeywell (Düsseldorf, Germany).
For spectrophotometric analyses, Merck (Darmstadt, Germany) provided the Folin–Ciocalteu reagent. Chlorogenic acid (5-caffeoylquinic acid), (±)-catechin hydrate, iron (II) sulfate heptahydrate, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), and iron (III) chloride hexahydrate were all supplied by Sigma-Aldrich (St. Louis, MO, USA).
For cellular assays, minimum essential medium (MEM), HEPES, trypsin–EDTA, antibiotic/antimycotic solution, NADH, SRB, sodium pyruvate, and trichloroaceric acid were all purchased from Sigma (St. Louis, MO, USA); fetal calf serum was supplied by Invitrogen Corporation (Carlsbad, CA, USA); Triton X-100 was purchased from Merck (Darmstadt, Germany); [1,2-³H(N)]-deoxy-D-glucose (³H-DG; 60 mCi/mmol) and ¹⁴C-D-fructose (¹⁴C-FRU; 250–360 mCi/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO, USA).
All other reagents were of analytical grade. Ultrapure water was obtained from a Direct-Pure UP Ultrapure & RO Lab Water System (RephiLe Bioscience Ltd., Boston, MA, USA).

Deionized water (DIW, <0.2
μS cm^–1, Ultrapur, Watrex) was used for the
preparation of all solutions. Formic acid (98%, p.a., Lach-Ner, Czech
Republic) was used for the formulation of photochemical media of various
molarities (M, i.e., mol L^–1). Sodium nitrate (99.99%,
Suprapur) from Merck was used as an additive to the photochemical
medium. Unless otherwise stated, both DIW and HCOOH were sub-boil
distilled. A commercial stock analytical standard solution of 1000
mg L^–1 Rh³⁺ (as RhCl₃) in
5% (w/w) HCl was obtained from Fluka. Specifications of additional
analytical standards, compounds used as potential metal ion mediators,
and other chemicals are given in the Supporting Information.

Inductively coupled plasma mass-spectrometry (ICP-MS) was performed as previously reported [38 (link)]. Briefly, samples were lyophilized and nitric acid (65% Suprapur, Merck; USA) was added for a 6-h digestion at RT. Samples were heated at 90 °C for 20 min followed by the addition of hydrogen peroxide (30% Aristart, BDH; UAE). Samples were left at room temperature for 30 min before heating again for a further 15 min at 70 °C. The average reduced volume was determined, and the samples were further diluted with 1% HNO₃ diluent. Measurements were made using an Agilent 7700 series ICP-MS instrument under routine multi-element operating conditions using a Helium Reaction Gas Cell. The instrument was calibrated using 0, 5, 10, 50, 100, and 500 ppb of certified multi-element ICP-MS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3, and ICP-MS-CAL-4, Accustandard; USA) for a range of elements. Certified internal standard solution containing 20 ppb of Yttrium (Y89) as an internal control (ICP-MS-IS-MIX1-1, Accustandard; USA) was used.

Assays for NH₄⁺, NO₂⁻, NO₃⁻, total dissolved nitrogen (TDN) and soluble reactive phosphorus (SRP)) from each sample were performed as described elsewhere^{26 (link)} by ion chromatography in an 861 Advance Compact IC system (Metrohm AG, Herisau, Switzerland). Ions were identified and quantified with internal and external standards prepared from Certified Standard Solutions (TraceCERT^®) (Merck). Chromatograms were done with the Metrohm IC Net 2.3 SR4 software.
Concentrations of Fe, Mg, S, Ca, Cl, Mn, K, Na, Al, As, B, Ba, Be, Bi, Co, Cu, Li, Mo, Ni, P, Pb, Rb, Sc, Se, Sr, V, Y, Zn, Zr, Cd, Cr, F, Ge and Ti were found out by inductively coupled plasma-mass spectrometry (ICP-MS) on a PerkinElmer ELAN9000 ICP-MS quadrupole spectrometer (Table 1). The method was previously described^{93 (link)}. Quantitative ICP-MS analysis was performed by an external standard calibration methodology.
The run was performed as follow: a blank (HNO₃ 1% (Suprapur^®, Merck), external standards with different concentrations, samples and a quality control standard at the end to control the instrumental drift and possible memory effects. All the solutions used (standards and samples) were acidified with nitric acid HNO₃ 1% (Suprapur^®, Merck). ¹⁰³Rh was added to the blank, standard, and sample solutions as an internal standard. External calibration and quality control standards were prepared from two initial multi-element solutions of 1000 mg L⁻¹: Multi-element Calibration Standard 2 (Perkin-Elmer) and ICP multi-element standard solution VI Certipur^® (Merck) containing all the analyzed elements.
Of all the chemical elements that were analyzed, only those represented in Table 1 (Fe, Mg, S, Ca, Cl, Mn, K and Na) presented concentrations high (≥3 ppb). The rest were scarce (<3 ppb) (Al, As, B, Ba, Be, Bi, Co, Cu, Li, Mo, Ni, P, Pb, Rb, Sc, Se, Sr, V, Y, Zn and Zr) or undetectable (F, Ge and Ti). Detection limits for constituents that were below detection limits ranged from 0.3 to 0.7 ppb.

C. krusei cells were grown on Sabouraud dextrose agar at 37°C for 24 hrs. Two to three fresh colonies were transferred to yeast peptone dextrose (YPD) broth (yeast extract 1%, peptone 2%, dextrose 2%), (Suprapur, Merck, Germany) at the same temperature for 48 hrs.
Total RNA was isolated from exponential-phase of the YPD broth cultures using RNeasy Mini kit (Qiagen) according to the manufacturer's instruction. Quantification of RNA was performed by absorbance at 260 nm using a Spectrophotometer (Biophotometer). The mean RNA concentration and the mean ratio for OD260/280 were 421+6 ng/µl and 1.8+0.04, respectively. For cDNA synthesis, 10 µl of total RNA was heated in 80°C for 10 min followed by cooling on ice. The master mixture contained 4 µl of 5x reverse transcriptase (RT) buffer, 10 mM of each dNTP, 20 pmol/µl random primer, 20 U RNase inhibitor (Fermentas, Burlington Canada), 200 U of Moloney Murine Leukemia Virus (MMuLV) Reverse Transcriptase (Fermentas, Burlington Canada), and 1.5 µl of DEPC-treated water. The cDNA synthesis was performed under following conditions: 42°C for 60 min, 70°C for 10 min, and finally cooling to 4°C. The integrity of cDNA was checked using the house keeping gene 18sRNA primers (as shown in table 2) which amplify region 1433–1639 (GB. EU348783.1). Samples with similar cDNA quality through 18sRNA PCR were stored at -70°C for further investigation.
PCR amplification of the ERG11 gene was conducted on samples using 1 µl of cDNA, specific forward and reverse primers corresponding to ERG11gene (Table 2), dNTP, MgCl, Taq DNA polymerase, and buffer (CinnaGen, Tehran, Iran). The thermocycling was performed using a Touch-Down amplification program on 2720 Thermal Cycler, ABI. The PCR condition was as the same as previously described (17 (link)).

Metal concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS). The prostate lobe and other organs [kidney, spleen, lung, liver and brain (left hemisphere)] were freeze-dried before being digested in 65% nitric acid (50-500 μL) (Suprapur, Merck, Bayswater, Australia; Cat#100441) overnight at room temperature and then at 90°C for 20 min. An equivalent volume of 30% hydrogen peroxide (VWR, Tingalpa, Australia; Cat#87003-224) was then added to each sample. Samples were incubated 30 min at room temperature and 15 min at 70°C and were further diluted with 1% nitric acid (900-1000 μl). Serum samples (50 μL) were diluted in 1% nitric acid (450 μL). Intracellular metal analysis of PrEC and TRAMP-C1 cells was performed as follows. Cellular pellets were generated as previously described [7 (link)] and to each pellet 50 μL of 65% nitric acid were added and the samples were incubated for 6 hours at room temperature before being heated at 90°C for 20 min. After digestion, 455 μL of 1% nitric acid were added to reach a final volume of 500 μL. All metal measurements were made using an Agilent 7700 series ICPMS instrument under routine multi-element operating conditions using a Helium Reaction Gas Cell. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 ppb of certified multi-element ICPMS standard calibration solutions (AccuStandard, New Haven, USA; Cat#ICP-MS-CAL2-1, ICP-MS-CAL-3 & ICP-MS-CAL-4) for a range of elements and a certified internal standard solution containing 200 ppb of Yttrium (Y89) was used as an internal control (AccuStandard; Cat#ICP-MS-IS-MIX1-1). The raw ppb values obtained were converted into either μg/g of wet weight for tissues (μg/g), to μmol/L for serum, or to ng/10⁶ cells for tissue culture as previously described [26 (link)].