Urine samples were collected over the course of the postprandial period following a morning void. At the end of the postprandial period, the collections were pooled and the specific gravity and volumes recorded. Urine was aliquoted, frozen, and stored for nitrogen content analysis. Total nitrogen was determined using the Dumas combustion method (Rapid N Exceed; Elementar Americas Inc., Mt. Laurel, NJ, USA).
Rapid n exceed
Manufactured by Elementar
Sourced in Germany, United States
The Rapid N exceed is a laboratory instrument designed for the determination of total nitrogen in solid and liquid samples. It utilizes the Dumas combustion method to analyze the samples and provide accurate nitrogen content measurements.
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15 protocols using rapid n exceed
1
Postprandial Urine Nitrogen Analysis
2
Urine Analysis for Nitrogen Content
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Urine samples were pooled by time periods (morning and afternoon) and volumes were recorded. Specific gravity, protein content, and refractivity index were measured (Reichert, Inc. Depew, NY). Urine was aliquoted, frozen and stored for later nitrogen analysis after each collection period. Total nitrogen was determined using Dumas combustion method (rapid N Exceed; Elementar Americas Inc., Mt. Laurel, NJ).
3
Characterizing Plant Mineral Profiles
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In experiment 1 (germination and seedling establishment) plants were too small for mineral analysis and only the fresh weight of shoots was recorded. For Experiment 2, plants were harvested, separated into shoots and roots, washed with tap water, followed by deionized water and blotted dry with paper towels and bagged to be dried in a forced-air oven at 70 °C until stable dry weight. Dry weight was recorded for shoots and roots. Samples were then ground in a Wiley mill to pass a 20-mesh (0.84 mm) screen. Tissue mineral concentration was based on shoot or root dry weight. Chloride was determined from nitric/acetic acid extracts by amperometric titration. The concentrations of tissue Na, P, K, Ca, Mg, and total-S, and of the micronutrients Fe, Cu, Mn, Zn, and Mo were determined from nitric acid digestions (Milestone, Ethos EZ Microwave Digestion, Shelton, CT, USA) of the dried, ground, plant material by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES, 3300DV, Perkin-Elmer Corp., Waltham, MA, USA). Nitrogen was determined by combustion in a Rapid N Exceed® analyzer (rNex, Elementar Americas Inc., Ronkonkoma, New York, NY, USA).
4
Protein Analysis Using Combustion Nitrogen
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A combustion nitrogen analyzer, rapid N exceed® (Elementar, Ronkonkoma, New York, NY, USA), was used for the protein analysis. In total, 250 mg aspartic acid was used as the blank and triplicates of 250 mg sample were loaded into the equipment. The nitrogen to protein factor was 6.25.
5
Proximate Composition Analysis of Fish
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The experimental diets and whole fish were analyzed following the AOAC protocols (2016) [15 ]. The moisture content of the samples was analyzed using the direct drying method. The samples were put in an oven at 105 ± 0.5 °C for 3 h until they reached a constant weight. The protein content of the whole fish was determined using automatic rapid nitrogen fixing (rapid N exceed, Elementar, Langenselbold, Germany). The ash content was determined by complete combustion of samples. Each sample (2.0 ± 0.5 g) was then ignited in the muffle furnace to ensure thorough combustion (600 °C, 2 h). The crude lipid content was determined using the Soxhlet extraction method with a Soxhlet extraction system (Extraction system-811, BUCHI, Flawil, Switzerland) and petroleum ether as the extraction solvent. The extraction process lasted for 10 h to ensure complete extraction of the lipid content from the sample. The gross energy content was determined through the utilization of a bomb calorimeter (LRY-600A, produced by Chuangxin Ltd., Hebei, China).
6
Protein Content Determination of WSF
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Protein content of 150 mg of WSF was determined by the Dumas method using a Rapid N Exceed bench top nitrogen analyser (Elementar, USA). Conversion factor was 6.25. Protein content was determined in triplicate.
7
Characterization of Maize Endosperm Composition
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Twenty kernels selected from the middle of each ear had their embryo, seed coat and aleurone removed. The resulting endosperms were dried in an oven (42 °C), and then ground into fine powder with a prototype machine (60HZ, 60S). The powder was filtered through an 80-mesh sieve to prepare samples for starch measurement.
For analysis of zeins in immature kernels, endosperms were collected at 18 and 24 DAP, and ground in the lipid nitrogen into fine powder. The extraction and analysis of zein proteins was according to the standard method of our laboratory42 (link). For assaying the total protein content, 60 mg of endosperm powder was used for measurement by the instrument of ELEMENTAR Rapid N exceed. The starch content of endosperm was measured with a Total Starch Assay Kit (K-TSTA; Megazyme) following the standard protocol43 (link). Amylose was quantified according to the Megazyme amylose/amylopectin assay procedure (K-AMYL; Megazyme). Six biological replicates were performed.
For analysis of zeins in immature kernels, endosperms were collected at 18 and 24 DAP, and ground in the lipid nitrogen into fine powder. The extraction and analysis of zein proteins was according to the standard method of our laboratory42 (link). For assaying the total protein content, 60 mg of endosperm powder was used for measurement by the instrument of ELEMENTAR Rapid N exceed. The starch content of endosperm was measured with a Total Starch Assay Kit (K-TSTA; Megazyme) following the standard protocol43 (link). Amylose was quantified according to the Megazyme amylose/amylopectin assay procedure (K-AMYL; Megazyme). Six biological replicates were performed.
8
Determination of Shoot and Root Nutrients
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Sodium, K and P in shoot and root samples were determined after digestion in a microwave (MARS 6, CEM Corporation, Matthews, NC, USA). Briefly: 500 mg of shoot or fruit was weighed into the digestion tube and subjected to element analyzed using ICP-OES (ICP-OES 5100, Agilent Technologies Inc., USA). Concentration of N was determined using the Dumas combustion method in an automatic elemental analyzer (Rapid N exceed, Elementar, Langenselbold, Germany).
9
Protein-Rich Seaweed Powder and Bioactive Compounds
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SP were prepared as previously reported (20 (link)). The dried SP powder had 92.8% protein content, as determined by rapid N exceed (Elementar, Langenselbold, Germany), with a nitrogen conversion factor of 6.25. OSAS (CAS#66829-29-6) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. EGCG (purity ≥ 98%) was purchased from Shanghai Macklin Biochemical Co., Ltd. All chemicals used were of analytical grade and used without further purification. Distilled water from a Lichun water purification system (Lichun, Jinan, China) was used in all the experiments.
10
Stable Isotope Analysis of Leaf Samples
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For experiment 1, total N was determined using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20–20 isotope ratio mass spectrometer (Sercon Ltd, Cheshire, United Kingdom) performed at UC Davies Stable Isotope Facility5 .
For experiment 3, 75–100 mg DW of homogenized leaf samples was used for the analysis with a rapid N exceed (Elementar, Germany). Aspartic acid (250 mg) was used as standard for calibration.
For experiment 3, 75–100 mg DW of homogenized leaf samples was used for the analysis with a rapid N exceed (Elementar, Germany). Aspartic acid (250 mg) was used as standard for calibration.
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