Unconjugated bilirubin were measured from serum samples, as described previously12 (link), using a high-performance liquid chromatograph (Merck, Hitachi, LaChrom, Vienna, Austria) equipped with a photodiode array detector (PDA, Shimadzu,) and a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm) and a phenomenex C18 HPLC guard column (4.0 × 3.0 mm). An isocratic mobile phase consisting of 0.1 M n-dioctylamine in methanol/water (95:5; v/v) and glacial acetic acid was used. Unconjugated bilirubin IX alpha (Frontier Scientific Europe, Carnforth, Lancashire, UK) served as an external standard. Sample and standard preparation and analysis were performed as previously published12 (link).
Potato dextrose agar (pda)
Manufactured by Shimadzu
Sourced in Austria, Japan
The PDA (Photodiode Array) is a laboratory equipment used for spectrophotometric analysis. It provides simultaneous detection of multiple wavelengths, enabling efficient and comprehensive analysis of samples.
Automatically generated - may contain errors
Lab products found in correlation
Fortis c18 hplc column, by Phenomenex (8 mentions)
C18 hplc guard column, by Phenomenex (4 mentions)
High performance liquid chromatography (hplc), by Merck Group (3 mentions)
Lc 20at, by Shimadzu (2 mentions)
Bilirubin alpha, by Merck Group (2 mentions)
Securityguard cartridges for c18 hplc columns, by Shimadzu (2 mentions)
Shim pack clc ods, by Shimadzu (1 mentions)
Pvdf membrane, by Pall Corporation (1 mentions)
Sphereclone, by Phenomenex (1 mentions)
B4126, by Merck Group (1 mentions)
C18 hplc column, by Phenomenex (1 mentions)
Esculin, by Merck Group (1 mentions)
Scopoletin, by Merck Group (1 mentions)
Umckalin, by Merck Group (1 mentions)
14 protocols using potato dextrose agar (pda)
1
Quantification of Unconjugated Bilirubin
2
HPLC Quantification of Unconjugated Bilirubin
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For a detailed analysis of UCB (isomers), the method of HPLC was applied (after)50 , as had been used and published by our group4 (link)51 (link) and others52 (link) previously. Briefly, fasting serum samples (stored light-protected in amber vials) were diluted in isocratic mobile phase (methanol, water, n-dioctylamine and acetic acid) and centrifuged. Supernatants were run on a chromatograph (Merck, Hitachi, LaChrom), equipped with a photodiode array detector (PDA, Shimadzu) and a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm), with a Phenomenex C18 HPLC guard column (4 × 3 mm). Sample preparation and analysis followed the previously published protocol4 (link). Unconjugated bilirubin (Frontier Scientific Europe, Carnforth, Lancashire, UK) served as an external standard/quality control. As an internal standard, a reference serum sample was run in each analysis.
3
HPLC Analysis of Intestinal SCFAs
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Intestinal contents were collected at the last day of the experiment. Each content was mixed with 1mL sterile 18 Ω de-ionized water. The pellet-water mixtures were homogenized by mixing at 3200 rpm for 5 minutes and centrifuged for 15 minutes at 13,000 rpm at 4°C. Supernatants were filtered using Acrodisc LC 13 mm sterile syringe filters with 0.2 μm PVDF membranes (Pall Life Sciences). The filtrates were used with short chain fatty acids (SCFAs) analyzed using high performance liquid chromatography (HPLC, LC-20AT, Shimadzu) technique, which equipped with a carbohydrate column (Aminex HPX-87H column, Biorad) and photodiode array detector (PDA, Shimadzu). The eluent was 5 mM H2SO4, fed at 0.6 mL/min, and column temperature set at 50°C. The run time was 60 minutes. Standard curves were generated by diluting 10 mM volatile fatty acid standard solution (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid) from 50 nM to 5000 nM with SCFAs concentrations normalized to soluble chemical oxygen demand.
4
HPLC-PDA Analysis of Compounds
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We modified a previous method,11 (link),12 (link) and used an HPLC system
equipped with a PDA (Shimadzu Corporation, Kyoto, Japan) to record ultraviolet
spectra from 200 to 400 nm. The column was a Shim-pack CLC-ODS (4.6 mm × 150 mm;
Shimadzu GLC Ltd., Tokyo, Japan), which was maintained at 40°C. Mobile phase A
was 20 mM ammonium formate-0.2% formic acid (1:1, v/v), and mobile phase B was
acetonitrile. The gradient conditions were 80% A and 20% B (0 min), 50% A and
50% B (20–25 min), and 80% A and 20% B (28–40 min). The flow rate was
1.0 mL/min, and the injection volume was 20 μL.
equipped with a PDA (Shimadzu Corporation, Kyoto, Japan) to record ultraviolet
spectra from 200 to 400 nm. The column was a Shim-pack CLC-ODS (4.6 mm × 150 mm;
Shimadzu GLC Ltd., Tokyo, Japan), which was maintained at 40°C. Mobile phase A
was 20 mM ammonium formate-0.2% formic acid (1:1, v/v), and mobile phase B was
acetonitrile. The gradient conditions were 80% A and 20% B (0 min), 50% A and
50% B (20–25 min), and 80% A and 20% B (28–40 min). The flow rate was
1.0 mL/min, and the injection volume was 20 μL.
5
HPLC-Based Quantification of Circulating UCB
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Circulating UCB levels were measured in serum/plasma samples following a well-established protocol [12 (link),27 (link)] using high-performance liquid chromatography (HPLC, Merck, Hitachi, LaChrom, Vienna, Austria), with a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm), Phenomenex SecurityGuard™ cartridges for the C18 HPLC columns (4 × 3 mm), and a photodiode array detector (PDA, Shimadzu). An isocratic mobile phase contained glacial acetic acid (6.01 g/L) and 0.1M n-dioctylamine in HPLC-grade methanol/water (96.5/3.5%). Before starting the procedure, all aliquots were centrifuged and a 50 μL sample was mixed with 200 μL of mobile phase. After a second centrifugation, 120 μL of the supernatant was injected into the HPLC at a flow rate of 1 mL/min.
Case-control pairs were analyzed in the same plate to minimize batch-to-batch fluctuation. Bilirubin (alpha) (purity≥ 98%, Sigma Aldrich) acted as an external standard (3.3% IIIα, 92.8% Ixα, and 3.9% XIIIα isomers, 450nm). One reference serum sample was assessed per analysis as an internal standard. The coefficient of variation (CV) between each plate was 3.76%.
Case-control pairs were analyzed in the same plate to minimize batch-to-batch fluctuation. Bilirubin (alpha) (purity≥ 98%, Sigma Aldrich) acted as an external standard (3.3% IIIα, 92.8% Ixα, and 3.9% XIIIα isomers, 450nm). One reference serum sample was assessed per analysis as an internal standard. The coefficient of variation (CV) between each plate was 3.76%.
6
Indirect Bilirubin Quantification by HPLC
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Indirect bilirubin levels (UCB) were measured in plasma samples by performing high-performance liquid chromatography analyses (HPLC; Merck, Hitachi, LaChrom) according to a widely-used protocol [28 (link),29 (link),35 (link)]. For the analyses, a Fortis C18 HPLC column (4.6 × 150 mm, 3 μm), cartridges (Phenomenex SecurityGuard™), and a photodiode array detector (PDA, Shimadzu) were used. All plasma samples were centrifuged and 50 μl sample was mixed with the isocratic mobile phase consisting of glacial acid (6.01 g/L) as well as 0.1 M n-dioctylamine solution in HPLC-grade methanol/water (96.5/3.5%). The samples were centrifuged repeatedly in order to inject 120 μl of the supernatant into the HPLC (flow rate: 1 mL/min). To reduce batch-to-batch fluctuation, all case-control pairs were analyzed using the same plate run and bilirubin α (purity≥98%, Sigma Aldrich) was used as the external standard.
7
Fecal SCFA Profiling via HPLC
8
Serum Bilirubin and Heme Analysis
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Serum samples for UCB measurement were stored in dark tubes, immediately after separation, as described previously37 (link). Unconjugated bilirubin and heme were measured in serum samples, using a high-performance liquid chromatograph (Merck, Hitachi, LaChrom, Vienna, Austria) equipped with a photodiode array detector (PDA, Shimadzu,) and a Fortis C18 HPLC column (4·6 × 150 mm, 3 μm) with a phenomenex C18 HPLC guard column (4·0 × 3·0 mm). Sample preparation and analysis were performed as previously published37 (link). Unconjugated bilirubin and haemin (both Frontier Scientific Europe, Carnforth, Lancashire, UK) served as external standards.
Free bilirubin was calculated from serum UCB and albumin levels, using a formula kindly provided by Dr. Silvia Gazzin and Dr. Claudio Tiribelli.
Free bilirubin was calculated from serum UCB and albumin levels, using a formula kindly provided by Dr. Silvia Gazzin and Dr. Claudio Tiribelli.
9
Quantification of Organic Acids via UFLC
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Organic acids were determined following a procedure previously optimized and described by the authors [10 ]. Analysis was performed on a Shimadzu 20A series ultra-fast liquid chromatograph (UFLC, Shimadzu Cooperation, Kyoto, Japan) coupled to photodiode array detector (PDA, Shimadzu), using 215 nm and 245 nm as the preferred wavelengths. Separation was achieved on a SphereClone (Phenomenex, Torrance, CA, USA) reverse phase C18 column (5 μm, 250 mm × 4.6 mm i.d) thermostatted at 35°C. Analytes were eluted with 3.6 mM sulphuric acid at a flow-rate of 0.8 mL/min. The organic acids found were quantified by comparison of the area of their peaks recorded at 215 nm or 245 nm (for ascorbic acid) with calibration curves obtained from commercial standards of each compound: oxalic acid (y = 1x107x + 96178; R2 = 0.999); quinic acid (y = 601768x + 8853.2; R2 = 1); malic acid (y = 952269x + 17803; R2 = 1); shikimic acid (y = 8x107 + 55079; R2 = 0.999). The results were expressed in mg per g of lyophilized decoction or infusion.
10
HPLC Analysis of UCB in EDTA Plasma
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UCB concentration in EDTA plasma was determined by high-performance liquid chromatography (HPLC) (Merck Hitachi LaChrom, Selm, Germany) as previously described by Wallner et al. (2012) [40 (link)]. A mobile phase consisting of 3.5% water and 96.5% methanol was used for the analysis. An amount of 50 µL of EDTA plasma was mixed with 200 µL mobile phase, then centrifuged for 10 min at a speed of 14,000 RPM and a temperature of 4 °C. An amount of 120 µL of the supernatant was pipetted into vials and placed in the autosampler. Analysis was performed using a Fortis C18 HPLC column (4.6 × 150 mm, 3 mm), a Phenomenex Security Guard cartridge for C18 HPLC columns (4 × 3 mm), and a photodiode array detector (PDA, Shimadzu, Tokyo, Japan). The bilirubin standard used was from Sigma Aldrich (St. Louis, MO, USA) (B4126).
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