Lc 10avp system

High-performance liquid chromatography (HPLC) analysis was performed on the LC-10Avp system (Shimadzu) equipped with a Supelco Discovery^® C18 column (5-μm particle size, 150-mm length × 4.6-mm internal diameter; Supelco); 0.1% phosphoric acid was used as the mobile phase; the flow rate was 0.6 ml/min; the detection wavelength was 280 nm; and the column temperature was 25°C.

WGP (5 mg/ml) was filtered via 0.45 μm microfiltration membrane. The filtered sample (20 μL) was loaded into a TSK-Gel G4000PWXL column (Tosoh, Shanghai branch, China) controlled by LC-10Avp system (Shimadzu, Shanghai branch, China). High performance gel permeation chromatography (HPGPC) was performed using 0.2 M NaCl as mobile phase at flow rate of 0.5 ml/min. T-series Dextran standards were used for reference standards.

Two milligrams of IOP‐A2 sample were dissolved in 0.2 M NaCl to a final concentration of 5 mg/mL. The solution was filtered through a 0.22 μm filter membrane, and 20 μL of the filtrate was analyzed by high‐performance gel permeation chromatography (HPGPC), using a LC‐10Avp system (Shimadzu Company) with a RID‐10A differential refraction detector and a TSK‐gel G‐3000 PWXL column (7.8 × 300 mm). The column temperature was 35°C, and the mobile phase was a 0.2 M NaCl solution used at a flow rate of 0.6 mL/min. A series of dextran with different molecular weights (1 kDa, 5 kDa, 12 kDa, 25 kDa, 50 kDa) was used to calibrate the chromatographic column and built a standard curve (Zhang, Liu, et al., 2019 (link)).

The measurement of plasma urea and calculation of eGFR were performed by the Sydney Adventist Hospital pathology laboratory using methods well established for clinical laboratories. Briefly, fasting plasma urea levels were determined by the enzymatic method on a Roche/Hitachi cobas c system. Estimated Glomerular filtration rate (eGFR) was calculated using the simplified Modification of Diet in Renal Disease study equation: GFR (ml/min/1.73m²) = 186 × (serum creatinine level [mg/dl])-1.154 × (age)-0.203× [0.742, if female] × [1.212, if black] [23 (link), 24 ].
Plasma levels of NAD⁺ and its metabolites [NADP⁺, cyclic ADP ribose (cADPR), nicotinamide (NAM), N-methylnicotinamide (MeNAM)] were measured by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), as previously described [25 ]. LC/MS/MS was carried out using a UPLC-MSD assembly consisting of an Accela UPLC pump, Accela AS injector, and a TSQ Vantage bench-top mass spectrometer (ThermoFisher Scientific, Waltham, US).
Plasma interleukin-6 (IL-6) levels were quantitated using the MILLIPLEX® MAP Human High Sensitivity T Cell Magnetic Bead Panel immunoassay (Merck KGaA, Darmstadt, Germany).
Plasma Kynurenine (Kyn) and Tryptophan (Trp) levels were measured by high-performance liquid chromatography (Shimadzu LC-10AVP system, equipped with SPD- 10A UV detector, Kyoto, Japan), as previously described [26 (link)].

The HPLC analysis was performed using a C18 column (150 × 4.6 mm i.d.: Luna^® 5 µm C18), Phenomenex, Torrance, CA, USA) with an LC-10Avp system (Shimadzu, Kyoto, Japan). The mobile phase comprises of acetonitrile (solvent A) and water (solvent B) (both contained 1% formic acid) at a flow rate of 0.4 mL·min⁻¹. The HPLC program was as follows: 10% to 15% B (v/v) for 5 min, 15% to 40% B from 5 to 15 min, 40% to 60% B from 20 to 22 min, and 10% B for 22 to 25 min. N, E, K, Q, DHK, DHQ, C and Af were monitored at 280 nm; p-CA and CA were monitored at 340 nm; and the anthocyanins were monitored at 530 nm. For further identification of the products, a liquid chromatography mass spectrum (LC-MS) system was used as previously described [19 (link)]. The quantitative products of Caffeic acid, Eriodictyol, Catechin, Quercetin and Dihydroquercetin were respectively used and their standard curves were plotted at 280 nm.

High-performance gel permeation chromatography (HPGPC) was used to assess weight-averaged molecular weights of the enzyme products with an LC-10Avp system (Shimadzu Company, Japan) and a TSK-gel G-3000 PWXL column (7.8 × 300 mm). The UPLC-MS method was performed using a Waters Acquity H-Class UPLC system linked to an ESI-MS mass spectrometer (amaZon speed ETD, Bruker, Germany) in the negative ion mode. Operating parameters were as follows: capillary voltage, 4.5 KV; capillary temperature, 200°C; nebulizer gas, 2 bar; dry gas, 6 l/min; scan range, 100–1000. RG oligosaccharide separation was carried out on an Acquity UPLC BEH Amide column (1.7 μm, 2.1 mm × 150 mm) at 35°C with a flow rate of 0.3 ml/min mobile phase. The mobile phase consisted of ACN and H₂O in a ratio of 20/80 (v/v) for mobile phase A, 80/20 (v/v) for mobile phase B, and pH 3.0 200 mM ammonium formate/50 mM formic acid buffer for mobile phase C. The run time for oligosaccharide separation was 60 min, and the elution procedure was as follows: concentration of C remained at 5% during the entire elution process for 0–30 min, 0%-20% A; 30–31 min, 20–35% A; 31–40 min, containing 35% A; 40–41 min, 35–0% A; 41–50 min, containing 0% A.

Basic procedures for the measurement of content of three chlorophyll precursors, protoporphyrin IX, Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethylester were followed by an HPLC-based procedure as described previously [38 (link)]. Extraction of chlorophyll precursors from seedlings was carried out using the same procedure as for chlorophyll extraction described above. The acetone extracts were applied to a HPLC system (LC10A VP system, Shimadzu) equipped with a reverse phase column (Symmetry C8, 150 mm × 4.6 mm, 3.5μm particle size, Waters, http://www.waters.com/) with a guard column (C8, 4.0 mm × 3.0 mm, Phenomenex, http://www.phenomenex.com/). Chlorophyll precursors were eluted by an anomalous gradient from solvent A (methanol:acetonitrile: 0.25 M pyridine = 50:25:25) to solvent B (methanol:acetonitrile:acetone = 20:60:20). Eluents were monitored with a fluorescence detector (RF-550, Shimadzu). For protoporphyrin IX detection, the excitation wavelength was set at 400 nm and fluorescence emission was detected at 634 nm, while for detections of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethylester, the excitation wavelength was set at 417 nm and fluorescence emission was detected at 600 nm. Content of these precursors was estimated through calibration curves determined from standard substances (Frontier Scientific, http://www.frontiersci.com/).