298 Analytical procedure Urinary content of 52Cr was quantified using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), which is a highly sensitive analytical method for measuring various metals, including chromium isotopes (Nexion 300X, Perkin Elmer, Medlon BV Enschede, The Netherlands).43 First, 24-h urine samples were 30-fold diluted with 0.5% HNO3 and 0.01% Triton X-100, after which 103Rh was added as internal standard (IS). Subsequently, the sample analyte was nebulized in a cyclonic spray chamber, atomized at extremely high temperature (ranging from 6,000-8,000oC) and ionized using argon (Ar) plasma. Using a vacuum of ~ 20 mL/min, the resulting ions were deflected into a quadrupole in which ions are separated based on their electrical charge and mass, under the influence of an alternating electrical current. Finally, chromium ions were detected via an electron multiplier. Urinary chromium (52Cr) was measured while applying the kinetic energy discrimination (KED) mode, ensuring a general reduction of polyatomic isobaric interferences (such as that of 40Ar12C). Final urinary 52Cr concentrations (nmol/L) were divided by urinary creatinine concentrations (mmol/L) as correction for hydration. Urinary creatinine levels were quantified by an enzymatic detection method following supplier’s instructions (Roche Diagnostics, Roche Modular P Analyzer, Mannheim, Germany). Quantification of fecal bacteria using fluorescent in-situ hybridization (FISH) The quantification of Faecalibacterium prausnitzii and Enterobacteriaceae was performed as previously described, with some minor modifications.44 Patients were asked to provide fecal samples at the time of the 24-h urine collection. Samples were mixed with 4.5 mL filtered phosphate-buffered saline (PBS) and centrifuged at 700 xg for 2-3 min. The resulting supernatant was 4-fold diluted with freshly-prepared 4% paraformaldehyde solution and stored overnight at 4°C. Before counting, samples were coded and randomized by an independent investigator. Serial dilutions were prepared to allow visual counting of total bacteria, Enterobacteriaceae and F. prausnitzii. Each dilution was spread over gelatin-coated glass slides and dried at room temperature. After addition of the appropriate bacterial probes (see Table 1), Eub338 (Rhodamine) for the total bacteria, Fprau645 (FITC) for F. prausnitzii and Ec1531 (CY3) for Enterobacteriaceae, slides were hybridized overnight at 50°C.45-47 In each glass slide well, 25 fields were manually quantified using fluorescent microscopes (Leica 2 or Olympus BH20 at 100X magnification). Bacteria were quantified using two different filters, FITC or CY3, based on the probe colour. Based on the absolute bacterial counts, the relative percentage of each bacterial group was calculated. Chapter 9
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